JP2024502360A - Novel steroid payloads, steroid linkers, containing ADCs, and uses thereof - Google Patents

Abstract

The present invention provides novel glucocorticosteroids, glucocorticosteroid linkers, and antibodies that bind to antigens expressed on immune cells, optionally antibodies or antibody fragments that bind to antigens expressed on human immune cells. Antibody drug conjugates (ADCs) are provided. In some cases, ADCs have short serum half-lives (≈24-72 hours or 24-48 hours or 12-24 hours or less in human VISTA knock-in rodents, or ≈1 to 24 hours in human or non-human primates). The anti-human VISTA (V-region immunoglobulin-containing suppressor of T cell activation (1)) antibody or anti-VISTA antigen-binding antibody fragment that binds to VISTA-expressing cells at physiological pH (3.5 days or less). In some cases, these ADCs have a rapid onset of action and are so effectively internalized by large numbers of immune cells that they are cleaved releasing large amounts of active steroid payload. The present invention also relates to the use of such ADCs and novel steroids for the treatment of autoimmune, allergic and inflammatory conditions.The present invention further relates to the use of such ADCs and novel steroids for the treatment of autoimmune, allergic and inflammatory conditions. By selectively delivering these anti-inflammatory agents to target immune cells such as neutrophils, B cells, T cells, Tregs, eosinophils, NK cells, macrophages, and myeloid cells, especially myeloid cells. The present invention relates to methods for reducing the adverse side effects and/or improving the effectiveness of glucocorticoid receptor agonists, thereby reducing their potential toxicity to non-target cells.

Description

The present invention provides novel glucocorticosteroids, glucocorticosteroid linkers, and antibodies that bind to antigens expressed on immune cells, typically human immune cells. Related to antibody drug conjugates (ADCs). In some embodiments, the ADC is an anti-human VISTA (V-region immunoglobulin-containing suppressor of T cell activation (1)) antibody or anti-VISTA antigen-binding antibody fragment, e.g., a human VISTA knock-in with a short serum half-life ( (24 to 27 hours or less) in rodents. The subject ADCs have a rapid onset of action and are effective for long periods of time because they are very effectively internalized by large numbers of immune cells, which are cleaved releasing large amounts of active steroid payload. . The invention also relates to the use of such ADCs and novel steroids for the treatment of autoimmune, allergic, inflammatory and cancer conditions, particularly acute and chronic autoimmune, allergic and inflammatory conditions. The present invention further describes the use of such ADCs to target immune cells, typically human immune cells, optionally monocytes, neutrophils, T cells, Tregs, eosinophils, B cells. selectively deliver these anti-inflammatory agents to either bone marrow cells, such as NK cells, or other immune cells involved in the pathology of the autoimmune, allergic, inflammatory, or cancerous condition being treated. The present invention relates to a method for reducing the harmful side effects and/or improving the efficacy of glucocorticoids by reducing their potential toxicity to non-target cells.

VISTA is an NCR ligand whose closest phylogenetic relative is PD-L1. VISTA is homologous to PD-L1 but exhibits a unique expression pattern that is restricted to the hematopoietic compartment. Specifically, VISTA is constitutively and highly expressed on CD11b ^high bone marrow cells and at lower levels on CD4 ⁺ and CD8 ⁺ T cells. Like PD-L1, VISTA is a highly immunosuppressive ligand, and like PD-L1, blocking VISTA allows the development of therapeutic immunity against cancer in preclinical tumor models. . While blocking VISTA improves immunity, particularly CD8 ⁺ and CD4 ⁺ -mediated T cell immunity, treatment with a soluble Ig fusion protein of the extracellular domain of VISTA (VISTA-Ig) improves immunity. It has been shown to suppress and prevent the progression of multiple mouse models of autoimmune diseases. Based on the foregoing, the use of antagonist anti-VISTA antibodies to promote T cell immunity and treat conditions where this is beneficial, such as cancer and infections, has been reported. Conversely, the use of agonist anti-VISTA antibodies to inhibit T cell immunity and treat conditions in which this is therapeutically beneficial, such as autoimmunity, allergies, and inflammatory conditions, has been reported. Unfortunately, some anti-VISTA antibodies, including some used in human clinical trials, require very frequent dosing, which is both inconvenient and ineffective for patients, such as cancer or autoimmune diseases. have a very short serum half-life, which is generally undesirable in the setting of treating chronic conditions. In addition, the potential use of anti-VISTA antibodies and VISTA fusion proteins to deliver payloads such as chemotherapeutic agents to cancer cells or tumor sites has been proposed.

Synthetic glucocorticoid receptor agonists (e.g., dexamethasone, prednisolone, budesonide, beclomethasone, betamethasone, cortisol, cortisone acetate, 16-alpha hydroxyprednisolone, dexamethasone, difluorazone, flumethasone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, Methylprednisolone, prednisone, prednisolone, mometasone, triamcinolone acetonide, etc.) are a powerful class of small molecules used in the treatment of inflammation and related disorders. These compounds are highly effective in inhibiting inflammation associated with different conditions such as autoimmune, allergic, and inflammatory disorders, cancer, and infectious diseases; Their usefulness in chronic treatment of is limited due to their severe side effects.

Based on the foregoing, several approaches have been explored to preserve the anti-inflammatory effects of synthetic glucocorticoids while eliminating unnecessary toxicity (Rosen, J and Miner, J N Endocrine Reviews 26:452-64). 2005)). In particular, antibody drug conjugates (ADCs) have been developed in which such compounds are conjugated to antibodies that target antigens expressed by immune cells, including CD40, CD163, CD74, PRLR, and TNF. . Nevertheless, in the field of autoimmune, allergic, and inflammatory diseases, improved anti-inflammatory, autoimmune, and allergy therapies, as well as compared to existing therapeutics for the treatment of such conditions, e.g. Therefore, there remains a need for the development of improved anti-inflammatory, autoimmune, and allergy therapeutics with increased efficacy, long-term efficacy, and/or reduced side effects.

Antibodies or antibody fragments that target antigens expressed by immune cells, typically human immune cells, in some embodiments anti-human VISTA, where the antibody or antibody fragment binds to VISTA-expressing cells at physiological pH. The present invention provides novel steroids, steroid linkers, and ADCs containing VISTA, which are antibodies or anti-human VISTA antibody fragments, to provide therapeutic agents for treating or preventing inflammation and disorders related thereto. This is the object of the invention.

Human VISTA knock-in herein as 1-72 hours, 1-32 hours, 1-16 hours, 1-8 hours, 1-4 hours, or 1-2 hours in rodents, or ≈3-4 days or less in cynomolgus monkeys. It is an object of the present invention to provide novel antibody-drug conjugates (ADCs) comprising anti-VISTA antibodies or antibody fragments that possess a very short serum half-life at physiological conditions (≈pH 7.5), as defined in It is a specific object of the present invention that the anti-VISTA antibody or antibody fragment is conjugated to a glucocorticoid receptor agonist or glucocorticoid receptor agonist linker as disclosed herein.

As demonstrated below, the subject ADC possesses a unique combination of advantages over previous ADCs for targeting and directing the internalization of anti-inflammatory agents, particularly steroids, into immune cells. , this is due to the novel nature of the steroid linker therein, which provides rapid internalization and release of large amounts of active payload once internalized by immune cells.

The subject ADCs also provide a high drug-antibody ratio (DAR) due to a reduced propensity for aggregation compared to previous ADCs containing glucocorticosteroids.

Additionally, the subject ADCs are more effectively internalized and release a more active glucocorticosteroid payload into target immune cells compared to previous ADCs containing glucocorticosteroids. provides high efficacy even at lower DAR.

In some embodiments, the subject ADCs also have steroid linker payloads and anti-VISTA antibodies or antibody fragments disclosed herein, particularly those that bind to VISTA-expressing immune cells at physiological pH and have a very short pK. Holds a compound interest in holdings. In particular, these ADCs, for example, bind to VISTA-expressing immune cells at very high densities and are effective for long periods of time (inducing anti-inflammatory activity) despite their very short PK. (i.e. have a PD much longer than their pK) and are therefore well suited for treating chronic inflammatory or autoimmune or allergic diseases, where long-term and repeated administration is therapeutically warranted. .

The subject ADCs comprising anti-VISTA antibodies or antibody fragments also target activated and non-activated T cells, Tregs, CD4 T cells, CD8 T cells, neutrophils, myeloid cells, monocytes, macrophages, eosinophils, Targeting a wide range of immune cells, including dendritic cells, NK cells, and endothelial cells, such ADCs are therefore useful for treating inflammatory diseases, diseases that involve any or all of these types of immune cells. or may be used to treat autoimmune or allergic diseases. However, alternatively, the subject ADCs may include antibodies or antibody fragments that bind other immune cell antigens, preferably antibodies or antibody fragments that effectively internalize target immune cells.

The subject ADCs have a rapid onset of efficacy and therefore can be used to treat for acute treatment. In the case of VISTA antibody-containing ADCs, these ADCs do not bind to B cells and therefore should not be immunosuppressive like free steroids.

In addition, in the case of VISTA antibody-containing ADCs, the subject ADCs act on Tregs, which are important immune cells involved in steroid efficacy, and inhibit resting and myeloid cells, monocytes, eosinophils, Tregs, CD8 T cells, It acts on both CD4 T cells, immune cells, etc. and is therefore active in both the active and remission phases of inflammatory and autoimmune conditions (inducing anti-inflammatory activity).

Also, in the case of VISTA antibody-containing ADCs, the subject ADCs act on neutrophils, immune cells that are essential for acute inflammation.

Also, in the case of VISTA antibody-containing ADCs, due to the high VISTA cell surface turnover, the subject ADCs internalize immune cells very rapidly and constitutively.

Furthermore, in the case of VISTA antibody-containing ADCs, the subject ADCs possess very short half-lives (PK) and selectively target immune cells, and therefore the subject ADCs are free from non-target cell-associated toxicity and undesirable There should be no tendency for peripheral steroid exposure (low non-specific loss effects).

Also, in the case of some VISTA antibody-containing ADCs according to the invention, since the anti-VISTA antibody is one that carries within it a silent IgG that does not induce immunological functions (no blockade of any VISTA biology). , the biological activity (anti-inflammatory effect) of the subject ADCs is entirely due to the anti-inflammatory payload (steroid).

Ｘが、フェニル、スピロ［３．３］ヘプタン、３～６員複素環、シクロアルキル、スピロアルキル、スピロヘテロシクロアルキル、二環式アルキル、ヘテロ二環式アルキル、［１．１．１］ビシクロペンタン、ビシクロ［２．２．２］オクタン、アダマンタン、及びキュバンから選択され、これらの各々を、独立して、Ｆ、Ｃｌ、Ｂｒ、Ｉ、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換することができ、その環構造の各々が、Ｎ、Ｓ、及びＯから選択される少なくとも１つの骨格ヘテロ原子を含み得、任意選択的に、１～４個のＣ１～３アルキルまたはＣ１～３ペルフルオロアルキルで更に置換され、
Ｚが、フェニル、スピロ［３．３］ヘプタン、３～６員複素環、シクロアルキル、スピロアルキル、スピロヘテロシクロアルキル、二環式アルキル、ヘテロ二環式アルキル、［１．１．１］ビシクロペンタン、ビシクロ［２．２．２］オクタン、アダマンタン、及びキュバンから選択され、これらの各々を、独立して、Ｆ、Ｃｌ、Ｂｒ、Ｉ、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換することができ、その環構造の各々が、Ｎ、Ｓ、及びＯから選択される少なくとも１つの骨格ヘテロ原子を含み得、任意選択的に、１～４個のＣ１～３アルキルまたはＣ１～３ペルフルオロアルキルで更に置換され、
Ｙが、ＣＨＲ１、Ｏ、Ｓ、及びＮＲ１から選択され、
Ｅが、ＣＨ２及びＯから選択され、
Ｇが、ＣＨ及びＮから選択され、
更に、ＧがＣＨであり、Ｘがフェニルであるときに、Ｚがフェニルではなく、
ＸへのＧの結合が、任意選択的にＣ１～３アルキル及びエチレンオキシドから選択され得、これらの各々が、独立して、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換され得、任意選択的に、１～４個のＣ１～３アルキルで更に置換され、
ＺへのＸの結合が、Ｘ及びＺ上の任意の利用可能な位置を占有し得、
置換基ＮＲ１Ｒ２が、Ｚ上の任意の利用可能な位置を占有し得、
Ｒ１が、Ｈ、１～８個の炭素の直鎖または分岐アルキル、アリール、及びヘテロアリール基から選択され、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｒ１がＨであるとき、Ｒ２が、Ｈ、１～８個の炭素の直鎖または分岐アルキル、アリール、及びヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｒ１がＨ、１～８個の炭素の直鎖もしくは分岐アルキル、またはヘテロアリールであるとき、Ｒ２が、
［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］ｍ－［Ｃ＝Ｏ］－［Ｖ］ｋ－Ｊ、
Ｃ＝Ｏ）ＯＣＨ２－ｐ－アミノフェニル－Ｎ－Ｖ－Ｊ、
（Ｃ＝Ｏ）ＯＣＨ２－ｐ－アミノフェニル－Ｎ－［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］ｍ－［Ｃ＝Ｏ］－［Ｖ］ｋ－Ｊ、及び
［Ｖ］ｋ－（Ｃ＝Ｏ）ＯＣＨ２－ｐ－アミノフェニル－Ｎ－［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］ｍ－［Ｃ＝Ｏ］－Ｊ、から選択される官能基であり得、
式中、ｍ＝１～６であり、ｋ＝０～１であり、Ｗの各順列が、独立して、Ｈ、ｎ＝１～４である［（ＣＨ２）ｎＲ３］、Ｒ３で終端する分岐アルキル鎖、及び１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基から選択され得、
Ｒ３が、Ｈ、メチル、エチル、イソプロピル、ＯＨ、Ｏ－アルキル、ＮＨ２、ＮＨ－アルキル、Ｎ－ジアルキル、ＳＨ、Ｓ－アルキル、グアニジン、尿素、カルボン酸、カルボキサミド、カルボン酸エステル、置換または非置換アリール、置換または非置換ヘテロアリールから選択され、当該アリール及びヘテロアリール置換基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択され得、
Ｖが、１～８個の炭素のアルキル鎖、１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基、１～８個の炭素を含む直鎖または分岐アルキル基、－Ｏ－アルキル、カルボン酸、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－、１～３アミノ酸配列であって、各アミノ酸が、独立して、Ｇｌｕ、Ｇｌｙ、Ａｓｎ、Ａｓｐ、Ｇｌｎ、Ｌｅｕ、Ｌｙｓ、Ａｌａ、βＡｌａ、Ｐｈｅ、Ｖａｌ、及びＣｉｔから選択される、１～３アミノ酸配列、アリール、ならびにヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｊが、－ＮＨ２、Ｎ３、チオ、シクロオクチン、－ＯＨ、－ＣＯ２Ｈ、トランスシクロオクテン、アルキニル、プロパルギル、

から選択される反応基であり、
式中、Ｒ３２が、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ３３が、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ３４が、Ｈ、Ｍｅ、テトラジン－Ｈ、及びテトラジン－Ｍｅであり、
Ｒ５が、－ＣＨ２ＯＨ、－ＣＨ２ＳＨ、－ＣＨ２Ｃｌ、－ＳＣＨ２Ｃｌ、－ＳＣＨ２Ｆ、－ＳＣＨ２ＣＦ３、ヒドロキシ、－ＯＣＨ２ＣＮ、－ＯＣＨ２Ｃｌ、－ＯＣＨ２Ｆ、－ＯＣＨ３、－ＯＣＨ２ＣＨ３、－ＳＣＨ２ＣＮ、及び

からなる群から選択され、
Ｒ６及びＲ７が、独立して、水素及びＣ１～１０アルキルから選択され、
Ｑが、Ｈ、

Ｒ８が１～８個の炭素の直鎖もしくは分岐アルキルである、Ｃ（Ｏ）Ｒ８、またはｎ＝１～４であり、Ｒ４＝Ｈ、アルキルもしくは分岐アルキル、またはＰ（Ｏ）ＯＲ４である、（Ｃ＝Ｏ）ＮＲ４ＣＨｎＮＲ４（Ｃ＝Ｏ）ＯＣＨ２－（Ｖ）ｎ－Ｊであり得、
Ａ１及びＡ２が、独立して、Ｈ及びＦから選択され、
別途明記しない限り、全ての可能な立体異性体が特許請求される、新規グルココルチコイドアゴニスト化合物を提供することが、本発明の具体的な目的である。 A novel glucocorticoid agonist compound of formula (I) having the following structure,

X is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C1 to further substituted with 3 alkyl or C1-3 perfluoroalkyl,
Z is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C1 to further substituted with 3 alkyl or C1-3 perfluoroalkyl,
Y is selected from CHR1, O, S, and NR1;
E is selected from CH2 and O;
G is selected from CH and N;
Furthermore, when G is CH and X is phenyl, Z is not phenyl,
The bond of G to optionally further substituted with 1 to 4 C1-3 alkyl,
the bond of X to Z may occupy any available position on X and Z;
The substituent NR1R2 may occupy any available position on Z;
R1 is selected from H, linear or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and the aryl and heteroaryl groups are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH , -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O) -, and dialkylamino C(O)-,
When R1 is H, R2 may be selected from H, straight chain or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and the aryl and heteroaryl groups are alkyl, haloalkyl, halogen, Biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O- , alkylamino-C(O)-, and dialkylamino-C(O)-,
When R1 is H, linear or branched alkyl of 1 to 8 carbons, or heteroaryl, R2 is
[(C=O)CH(W)NH]m-[C=O]-[V]kJ,
C=O)OCH2-p-aminophenyl-NVJ,
(C=O)OCH2-p-aminophenyl-N-[(C=O)CH(W)NH]m-[C=O]-[V]k-J, and [V]k-(C= O) OCH2-p-aminophenyl-N-[(C=O)CH(W)NH]m-[C=O]-J,
where m=1 to 6, k=0 to 1, and each permutation of W is independently H, n=1 to 4 [(CH2)nR3], a branch terminating in R3 may be selected from alkyl chains, and linear or branched polyethylene oxide groups containing from 1 to 13 units;
R3 is H, methyl, ethyl, isopropyl, OH, O-alkyl, NH2, NH-alkyl, N-dialkyl, SH, S-alkyl, guanidine, urea, carboxylic acid, carboxamide, carboxylic acid ester, substituted or unsubstituted selected from aryl, substituted or unsubstituted heteroaryl, wherein the aryl and heteroaryl substituents are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, may be selected from thiols, thioalkyls, guanidines, ureas, carboxylic acids, alkoxyls, carboxamides, carboxylic esters, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein the aryl and The heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl- may be substituted with a functional group selected from C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH2, N3, thio, cyclooctyne, -OH, -CO2H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R32 is selected from Cl, Br, F, mesylate, and tosylate, and R33 is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-(4- nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, R34 is H, Me, tetrazine-H, and tetrazine-Me;
R5 is -CH2OH, -CH2SH, -CH2Cl, -SCH2Cl, -SCH2F, -SCH2CF3, hydroxy, -OCH2CN, -OCH2Cl, -OCH2F, -OCH3, -OCH2CH3, -SCH2CN, and

selected from the group consisting of
R6 and R7 are independently selected from hydrogen and C1-10 alkyl;
Q is H,

R8 is straight chain or branched alkyl of 1 to 8 carbons, C(O)R8, or n=1 to 4 and R4=H, alkyl or branched alkyl, or P(O)OR4; (C=O)NR4CHnNR4(C=O)OCH2-(V)n-J;
A1 and A2 are independently selected from H and F;
It is a specific object of the present invention to provide novel glucocorticoid agonist compounds, in which all possible stereoisomers are claimed, unless otherwise stated.

X and Z are independently selected from phenyl, spiro[3.3]heptane, [1.1.1]bicyclopentane, and bicyclo[2.2.2]octane, and Y is CH ₂ and O and the permutations of W are independently selected from CH ₂ CH ₂ CO ₂ H and H, and further, when G is CH and X is phenyl, Z is not phenyl. It is a specific object of the present invention to provide glucocorticoid agonist compounds as described.

A glucocorticoid agonist compound as described above, selected from any of the glucocorticoid agonist compounds disclosed in Example 3, except INX J and INX L, or selected from those shown in FIG. It is a specific object of the present invention to provide.

The present invention provides glucocorticoid agonist compounds as described above, selected from INX-steroid payloads, INX-steroid linkers, and INX-antibody drug conjugate (ADC) compounds, excluding INX J and INX L. This is the specific purpose of

から選択される、前述に記載のグルココルチコイドアゴニスト化合物を提供することが、本発明の具体的な目的である。 below:

It is a specific object of the present invention to provide glucocorticoid agonist compounds as described above, selected from:

Provided is a glucocorticoid agonist compound as described above, attached directly or indirectly to at least one cleavable or non-cleavable peptide and/or non-peptide linker (i.e., a "steroid-linker payload"). is a specific objective of the present invention.

at least one cleavable or non-cleavable linker ("L"), optionally a "heterobifunctional group" that is a chemical moiety used to connect a linker in a compound to an antibody or antibody fragment; A compound (steroid-linker payload) comprising a "heterotrifunctional group""Q" and at least one anti-inflammatory agent ("AI"), wherein "AI" has the following structure:
Q-L-AI or AI-L-Q
It is a specific object of the present invention to provide a compound (steroid-linker payload) which is a glucocorticoid agonist compound according to any of the foregoing which may be represented by:

It is a specific object of the present invention to provide a steroid-linker payload as described above, wherein the linker is selected from those disclosed herein.

PAB and/or amino acids or peptides, optionally 1 to 12 amino acids, more optionally dipeptides, tripeptides, quatrapeptides, pentapeptides, more optionally Gly, Asn, Asp, Gln, Leu, Lys, Ala, Phe, Cit, Val, Val-Cit, Val-Ala, Val-Gly, Val-Gln, Ala-Val, Cit-Cit, Lys-Val-Cit, Asp-Val-Ala, Ala-Ala-Asn, Asp-Val-Ala, Ala-Val-Cit, Ala-Asn-Val, βAla-Leu-Ala-Leu, Lys-Val-Ala, Val-Leu-Lys, Asp-Val-Cit, at least one cleavable or non-cleavable linker selected from Val-Ala-Val, and Ala-Ala-Asn, or optionally at least one of GlcA, PAB, and Glu-Gly. It is a specific object of the present invention to provide a steroid-linker payload according to any of the foregoing.

It is an embodiment of the present invention to provide a steroid-linker payload as described above comprising at least one cleavable linker and/or a sacrificial linker attached directly or indirectly to a glucocorticoid agonist steroid compound. purpose.

selected from any of the glucocorticoid agonist compounds or steroid-linker payload compounds disclosed in Examples, e.g., Example 3, except INX J and INX L, and the compounds described in Figures 118A-O. , a glucocorticoid agonist compound or a steroid-linker payload compound as described in any of the foregoing.

below:
(i) INX-SM-3-GluGly-alkoxyamine, INX-SM-4-GluGly-alkoxyamine, INX-SM-53-GluGly-alkoxyamine, INX-SM-54-GluGly-alkoxyamine, INX-SM -56-GluGly-alkoxyamine, INX-SM-98-GluGly-alkoxyamine, INX-SM-6-GluGly-alkoxyamine, INX-SM-2-GluGly-alkoxyamine, INX-SM-57-GluGly-alkoxy Amine, INX-SM-31-GluGly-alkoxyamine, INX-SM-32-GluGly-alkoxyamine, INX-SM-10-GluGly-alkoxyamine, INX-SM-40-GluGly-alkoxyamine, INX-SM- 34-GluGly-alkoxyamine, INX-SM-28-GluGly-alkoxyamine, INX-SM-27-GluGly-alkoxyamine, INX-SM-35-GluGly-alkoxyamine, INX-SM-8-GluGly-alkoxyamine , INX-SM-7-GluGly-alkoxyamine, INX-SM-33-GluGly-alkoxyamine, or Glu-Gly is replaced with a different cleavable peptide linker and another INX or INX-SM payload is shown in FIG. or (ii) INX-SM-53-GluGly- Bromoacetyl, INX-SM-3-GluGly-bromoacetyl, INX-SM-54-GluGly-bromoacetyl, INX-SM-1-GluGly-bromoacetyl, INX-SM-4-GluGly-bromoacetyl, INX-SM -2-GluGly-bromoacetyl, INX-SM-47-GluGly-bromoacetyl, INX-SM-7-GluGly-bromoacetyl, INX-SM-8-GluGly-bromoacetyl, INX-SM-56-GluGly-bromo Acetyl, INX-SM-32-GluGly-bromoacetyl, INX-SM-6-GluGly-bromoacetyl, INX-SM-10-GluGly-bromoacetyl, INX-SM-33-GluGly-bromoacetyl, INX-SM- 31-GluGly-bromoacetyl, INX-SM-35-GluGly-bromoacetyl, INX-SM-9-GluGly-bromoacetyl, INX-SM-28-GluGly-bromoacetyl, INX-SM-27-GluGly-bromoacetyl , INX-SM-34-GluGly-bromoacetyl, INX-SM-35-GluGly-bromoacetyl, INX-SM-40-GluGly-bromoacetyl, or Glu-Gly is replaced with a different cleavable peptide linker, a glucocorticoid agonist (payload) linker conjugate in which another INX or INX-SM payload is substituted for the INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O;
(iii) INX-SM-53-GluGly-dibenzocyclooctyne, INX-SM-1-GluGly-dibenzocyclooctyne, INX-SM-4-GluGly-dibenzocyclooctyne, INX-SM-54-GluGly-dibenzocyclooctyne , INX-SM-7-GluGly-dibenzocyclooctyne, INX-SM-8-GluGly-dibenzocyclooctyne, INX-SM-2-GluGly-dibenzocyclooctyne, INX-SM-57-GluGly-dibenzocyclooctyne, INX -SM-40-GluGly-dibenzocyclooctyne, INX-SM-34-GluGly-dibenzocyclooctyne, INX-SM-28-GluGly-dibenzocyclooctyne, INX-SM-27-GluGly-dibenzocyclooctyne, INX-SM -35-GluGly-dibenzocyclooctyne, INX-SM-9-GluGly-dibenzocyclooctyne, INX-SM-10-GluGly-dibenzocyclooctyne, INX-SM-31-GluGly-dibenzocyclooctyne, INX-SM-32 -GluGly-dibenzocyclooctyne, INX-SM-33-GluGly-dibenzocyclooctyne, INX-SM-56-GluGly-dibenzocyclooctyne, INX-SM-6-GluGly-dibenzocyclooctyne, INX-SM-3-GluGly - an INX contained therein in which dibenzocyclooctyne, or GluGly, is replaced with a different cleavable peptide linker and another INX or INX-SM payload is optionally selected from those of Figures 118A-O. - a glucocorticoid agonist (payload) linker conjugate substituted with the SM payload, or (iv) INX-SM-1-GluGly-NHS ester, INX-SM-31-GluGly-NHS ester, INX-SM-32- GluGly-NHS ester, INX-SM-33-GluGly-NHS ester, INX-SM-53-GluGly-NHS ester, INX-SM-7-GluGly-NHS ester, INX-SM-8-GluGly-NHS ester, INX -SM-2-GluGly-NHS ester, INX-SM-56-GluGly-NHS ester, INX-SM-6-GluGly-NHS ester, INX-SM-54-GluGly-NHS ester, INX-SM-4-GluGly -NHS ester, INX-SM-53-GluGly-NHS ester, INX-SM-3-GluGly-NHS ester, INX-SM-9-GluGly-NHS ester, INX-SM-40-GluGly-NHS ester, INX- SM-34-GluGly-NHS ester, INX-SM-28-GluGly-NHS ester, INX-SM-34-GluGly-NHS ester, INX-SM-28-GluGly-NHS ester, INX-SM-27-GluGly- NHS ester, INX-SM-35-GluGly-NHS ester, INX-SM-10-GluGly-NHS ester, or GluGly is replaced with a different cleavable peptide linker and another INX or INX-SM payload is shown in FIG. a glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, optionally selected from those of 118A-O;
(v) INX-SM-1-GluGly-maleimide, INX-SM-3-GluGly-maleimide, INX-SM-4-GluGly-maleimide, INX-SM-8-GluGly-maleimide, INX-SM-2-GluGly -Maleimide, INX-SM-7-GluGly-maleimide, INX-SM-56-GluGly-maleimide, INX-SM-6-GluGly-maleimide, INX-SM-54-GluGly-maleimide, INX-SM-53-GluGly -Maleimide, INX-SM-33-GluGly-maleimide, INX-SM-35-GluGly-maleimide, INX-SM-40-GluGly-maleimide, INX-SM-34-GluGly-maleimide, INX-SM-28-GluGly -Maleimide, INX-SM-27-GluGly-maleimide, INX-SM-35-GluGly-maleimide, INX-SM-9-GluGly-maleimide, INX-SM-10-GluGly-maleimide, INX-SM-31-GluGly - maleimide, INX-SM-32-GluGly-maleimide, INX-SM-57-GluGly-maleimide, or GluGly is replaced with a different cleavable peptide linker and another INX or INX-SM payload is a glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, optionally selected from those of O, or (vi) INX-SM-3-GluGly-tetrazine; INX-SM-53-GluGly-tetrazine, INX-SM-1-GluGly-tetrazine, INX-SM-54-GluGly-tetrazine, INX-SM-6-GluGly-tetrazine, INX-SM-56-GluGly-tetrazine, INX-SM-4-GluGly-tetrazine, INX-SM-10-GluGly-tetrazine, INX-SM-31-GluGly-tetrazine, INX-SM-32-GluGly-tetrazine, INX-SM-33-GluGly-tetrazine, INX-SM-7-GluGly-tetrazine, INX-SM-8-GluGly-tetrazine, INX-SM-9-GluGly-tetrazine, INX-SM-27-GluGly-tetrazine, INX-SM-35-GluGly-tetrazine, INX-SM-2-GluGly-tetrazine, INX-SM-40-GluGly-tetrazine, INX-SM-34-GluGly-tetrazine, INX-SM-28-GluGly-tetrazine, INX-SM-27-GluGly-tetrazine, or GluGly is replaced with a different cleavable peptide linker and another INX or INX-SM payload is replaced with an INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O. or (vii) INX-SM-6-GluGly-amine, INX-SM-54-GluGly-amine, INX-SM-4-GluGly-amine, INX-SM -53-GluGly-amine, INX-SM-2-GluGly-amine, INX-SM-56-GluGly-amine, INX-SM-57-GluGly-amine, INX-SM-35-GluGly-amine, INX-SM -27-GluGly-amine, INX-SM-40-GluGly-amine, INX-SM-34-GluGly-amine, INX-SM-28-GluGly-amine, INX-SM-35-GluGly-amine, INX-SM -9-GluGly-amine, INX-SM-10-GluGly-amine, INX-SM-31-GluGly-amine, INX-SM-32-GluGly-amine, INX-SM-33-GluGly-amine, INX-SM -7-GluGly-amine, INX-SM-8-GluGly-amine, INX-SM-1-GluGly-amine, INX-SM-3-GluGly-amine, or GluGly is replaced with a different cleavable peptide linker. , a glucocorticoid agonist (payload) linker conjugate, in which another INX or INX-SM payload is substituted for an INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O; or (viii) INX-SM-53-PAB-GluGly-alkoxyamine, INX-SM-1-PAB-GluGly-alkoxyamine, INX-SM-3-PAB-GluGly-alkoxyamine, INX-SM-2-PAB -GluGly-alkoxyamine, INX-SM-56-PAB-GluGly-alkoxyamine, INX-SM-35-PAB-GluGly-alkoxyamine, INX-SM-25-PAB-GluGly-alkoxyamine, INX-SM-27 -PAB-GluGly-alkoxyamine, INX-SM-35-PAB-GluGly-alkoxyamine, INX-SM-9-PAB-GluGly-alkoxyamine, INX-SM-10-PAB-GluGly-alkoxyamine, INX-SM -31-PAB-GluGly-alkoxyamine, INX-SM-32-PAB-GluGly-alkoxyamine, INX-SM-33-PAB-GluGly-alkoxyamine, INX-SM-57-PAB-GluGly-alkoxyamine, INX -SM-7-PAB-GluGly-alkoxyamine, INX-SM-8-PAB-GluGly-alkoxyamine, INX-SM-6-PAB-GluGly-alkoxyamine, INX-SM-54-PAB-GluGly-alkoxyamine , INX-SM-4-PAB-GluGly-alkoxyamine, INX-SM-40-PAB-GluGly-alkoxyamine, INX-SM-34-PAB-GluGly-alkoxyamine, or GluGly and/or PAB are 118A-O, and another INX or INX-SM payload is substituted with the INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O. , another glucocorticoid agonist (payload) linker conjugate, or (ix) INX-SM-1-PAB-GluGly-bromoacetyl, INX-SM-3-PAB-GluGly-bromoacetyl, INX-SM-2-PAB -GluGly-bromoacetyl, INX-SM-7-PAB-GluGly-bromoacetyl, INX-SM-8-PAB-GluGly-bromoacetyl, INX-SM-40-PAB-GluGly-bromoacetyl, INX-SM-56 -PAB-GluGly-bromoacetyl, INX-SM-6-PAB-GluGly-bromoacetyl, INX-SM-154PAB-GluGly-bromoacetyl, INX-SM-4-PAB-GluGly-bromoacetyl, INX-SM-33 -PAB-GluGly-bromoacetyl, INX-PAB-GluGly-bromoacetyl, INX-SM-32-PAB-GluGly-bromoacetyl, INX-SM-10-PAB-GluGly-bromoacetyl, INX-SM-34-PAB -GluGly-bromoacetyl, INX-SM-31-PAB-GluGly-bromoacetyl, INX-SM-9-PAB-GluGly-bromoacetyl, INX-SM-28-PAB-GluGly-bromoacetyl, INX-SM-27 -PAB-GluGly-bromoacetyl, INX-SM-35-PAB-GluGly-bromoacetyl, INX-SM-53-PAB-GluGly-bromoacetyl, or GluGly and/or PAB with different cleavable peptide linkers or non-cleavable peptide linkers. Another glucocorticoid agonist is substituted with a peptide linker and another INX or INX-SM payload is substituted with the INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O. (payload) linker conjugate,
(x) INX-SM-6-PAB-GluGly-dibenzocyclooctyne, INX-SM-54-PAB-GluGly-dibenzocyclooctyne,
INX-SM-4-PAB-GluGly-dibenzocyclooctyne, INX-SM-53-PAB-GluGly-dibenzocyclooctyne, INX-SM-1-PAB-GluGly-dibenzocyclooctyne, INX-SM-7-PAB- GluGly-dibenzocyclooctyne, INX-SM-8-PAB-GluGly-dibenzocyclooctyne, INX-SM-2-PAB-GluGly-dibenzocyclooctyne, INX-SM-56-PAB-GluGly-dibenzocyclooctyne, INX- SM-57-PAB-GluGly-dibenzocyclooctyne, INX-SM-33-PAB-GluGly-dibenzocyclooctyne, INX-SM-32-PAB-GluGly-dibenzocyclooctyne, INX-SM-31-PAB-GluGly- Dibenzocyclooctyne, INX-SM-3-PAB-GluGly-dibenzocyclooctyne, INX-SM-9-PAB-GluGly-dibenzocyclooctyne, INX-SM-27-PAB-GluGly-dibenzocyclooctyne, INX-SM- 35-PAB-GluGly-dibenzocyclooctyne, INX-SM-34-PAB-GluGly-dibenzocyclooctyne, INX-SM-28-PAB-GluGly-dibenzocyclooctyne, INX-SM-40-PAB-GluGly-dibenzocyclooctyne Octyne, INX-SM-10-PAB-GluGly-dibenzocyclooctyne, or GluGly and/or PAB is replaced with a different cleavable peptide linker or non-peptide linker, and another INX or INX-SM payload is shown in FIG. 118A another glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, optionally selected from those of -GluGly-NHS ester, INX-SM-54-PAB-GluGly-NHS ester, INX-SM-4-PAB-GluGly-NHS ester, INX-SM-53-PAB-GluGly-NHS ester, INX-SM-1 -PAB-GluGly-NHS ester, INX-SM-3-PAB-GluGly-NHS ester, INX-SM-33-PAB-GluGly-NHS ester, INX-SM-57-PAB-GluGly-NHS ester, INX-SM -7-PAB-GluGly-NHS ester, INX-SM-8-PAB-GluGly-NHS ester, INX-SM-27-PAB-GluGly-NHS ester, INX-SM-35-PAB-GluGly-NHS ester, INX -SM-9-PAB-GluGly-NHS ester, INX-SM-10-PAB-GluGly-NHS ester, INX-SM-31-PAB-GluGly-NHS ester, INX-SM-32-PAB-GluGly-NHS ester , INX-SM-40-PAB-GluGly-NHS ester, INX-SM-34-PAB-GluGly-NHS ester, INX-SM-28-PAB-GluGly-NHS ester, INX-SM-2-PAB-GluGly- The NHS ester or GluGly and/or PAB is replaced with a different cleavable peptide or non-peptide linker, and another INX or INX-SM payload is optionally selected from those of FIGS. 118A-O. or (xii) INX-SM-1-PAB-GluGly-Maleimide, INX-SM-53-PAB- GluGly-maleimide, INX-SM-5-PAB-GluGly-maleimide, INX-SM-2-PAB-GluGly-maleimide, INX-SM-8-PAB-GluGly-maleimide, INX-SM-56-PAB-GluGly- maleimide, INX-SM-54-PAB-GluGly-maleimide, INX-SM-4-PAB-GluGly-maleimide, INX-SM-57-PAB-GluGly-maleimide, INX-SM-7-PAB-GluGly-maleimide, INX-SM-32-PAB-GluGly-maleimide, INX-SM-31-PAB-GluGly-maleimide, INX-SM-53-PAB-GluGly-maleimide, INX-SM-3-PAB-GluGly-maleimide, INX- SM-34-PAB-GluGly-maleimide, INX-SM-28-PAB-GluGly-maleimide, INX-SM-40-PAB-GluGly-maleimide, INX-SM-27-PAB-GluGly-maleimide, INX-SM- 35-PAB-GluGly-maleimide, INX-SM-9-PAB-GluGly-maleimide, INX-SM-10-PAB-GluGly-maleimide, or GluGly and/or PAB are different cleavable peptide or non-peptide linkers. and another glucocorticoid agonist (payload ) linker conjugate, or (xiii) INX-SM-6-PAB-GluGly-tetrazine, INX-SM-54-PAB-GluGly-tetrazine, INX-SM-4-PAB-GluGly-tetrazine, INX-SM-53 -PAB-GluGly-tetrazine, INX-SM-1-PAB-GluGly-tetrazine, INX-SM-3-PAB-GluGly-tetrazine, INX-SM-57-PAB-GluGly-tetrazine, INX-SM-7-PAB -GluGly-tetrazine, INX-SM-8-PAB-GluGly-tetrazine, INX-SM-2-PAB-GluGly-tetrazine, INX-SM-31-PAB-GluGly-tetrazine, INX-SM-32-PAB-GluGly -Tetrazine, INX-SM-33-PAB-GluGly-tetrazine, INX-SM-56-PAB-GluGly-tetrazine, INX-SM-35-PAB-GluGly-tetrazine, INX-SM-9-PAB-GluGly-tetrazine , INX-SM-40-PAB-GluGly-tetrazine, INX-SM-34-PAB-GluGly-tetrazine, INX-SM-28-PAB-GluGly-tetrazine, INX-SM-27-PAB-GluGly-tetrazine, INX - SM-35-PAB-GluGly-tetrazine, INX-SM-10-PAB-GluGly-tetrazine, or GluGly and/or PAB are replaced with a different cleavable peptide linker or non-peptide linker, and another INX or INX - another glucocorticoid agonist (payload) linker conjugate, in which the SM payload is replaced with an INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O, or (xiv) INX-SM-1-PAB-GluGly-amine, INX-SM-3-PAB-GluGly-amine, INX-SM-8-PAB-GluGly-amine, INX-SM-2-PAB-GluGly-amine, INX- SM-56-PAB-GluGly-amine, INX-SM-6-PAB-GluGly-amine, INX-SM-54-PAB-GluGly-amine, INX-SM-4-PAB-GluGly-amine, INX-SM- 53-PAB-GluGly-amine, INX-SM-33-PAB-GluGly-amine, INX-SM-53-PAB-GluGly-amine, INX-SM-7-PAB-GluGly-amine, INX-SM-9- PAB-GluGly-amine, INX-SM-35-PAB-GluGly-amine, INX-SM-40-PAB-GluGly-amine, INX-SM-34-PAB-GluGly-amine, INX-SM-28-PAB- GluGly-amine, INX-SM-27-PAB-GluGly-amine, INX-SM-35-PAB-GluGly-amine, INX-SM-10-PAB-GluGly-amine, INX-SM-31-PAB-GluGly- The amine, INX-SM-32-PAB-GluGly-amine, or GluGly and/or PAB is replaced with a different cleavable peptide linker or non-peptide linker, and another INX or INX-SM payload is shown in Figures 118A-O. another glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, optionally selected from (xv) INX-SM-1-PAB-GlcA -Alkoxyamine, INX-SM-35-PAB-GlcA-alkoxyamine, INX-SM-9-PAB-GlcA-alkoxyamine, INX-SM-10-PAB-GlcA-alkoxyamine, INX-SM-54-PAB -GlcA-alkoxyamine, INX-SM-31-PAB-GlcA-alkoxyamine, INX-SM-32-PAB-GlcA-alkoxyamine, INX-SM-33-PAB-GlcA-alkoxyamine, INX-SM-57 -PAB-GlcA-alkoxyamine, INX-SM-7-PAB-GlcA-alkoxyamine, INX-SM-8-PAB-GlcA-alkoxyamine, INX-SM-2-PAB-GlcA-alkoxyamine, INX-SM -56-PAB-GlcA-alkoxyamine, INX-SM-6-PAB-GlcA-alkoxyamine, INX-SM-4-PAB-GlcA-alkoxyamine, INX-SM-53-PAB-GlcA-alkoxyamine, INX -SM-27-PAB-GlcA-alkoxyamine, INX-SM-40-PAB-GlcA-alkoxyamine, INX-SM-34-PAB-GlcA-alkoxyamine, INX-SM-28-PAB-GlcA-alkoxyamine , INX-SM-3-PAB-GlcA-alkoxyamine, or GlcA and/or PAB is replaced with a different cleavable peptide or non-peptide linker and another INX or INX-SM payload is shown in FIGS. 118A-O another glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, optionally selected from -Bromoacetyl, INX-SM-4-PAB-GlcA-bromoacetyl, INX-SM-56-PAB-GlcA-bromoacetyl, INX-SM-54-PAB-GlcA-bromoacetyl, INX-SM-4-PAB -GlcA-bromoacetyl, INX-SM-53-PAB-GlcA-bromoacetyl, INX-SM-7-PAB-GlcA-bromoacetyl, INX-SM-8-PAB-GlcA-bromoacetyl, INX-SM-2 -PAB-GlcA-bromoacetyl, INX-SM-40-PAB-GlcA-bromoacetyl, INX-SM-57-PAB-GlcA-bromoacetyl, INX-SM-33-PAB-GlcA-bromoacetyl, INX-SM -10-PAB-GlcA-bromoacetyl, INX-SM-34-PAB-GlcA-bromoacetyl, INX-SM-31-PAB-GlcA-bromoacetyl, INX-SM-32-PAB-GlcA-bromoacetyl, INX -SM-35-PAB-GlcA-bromoacetyl, INX-SM-9-PAB-GlcA-bromoacetyl, INX-SM-28-PAB-GlcA-bromoacetyl, INX-SM-27-PAB-GlcA-bromoacetyl , INX-SM-1-PAB-GlcA-bromoacetyl, or GluGly and/or PAB is replaced with a different cleavable peptide or non-peptide linker, and another INX or INX-SM payload is shown in FIGS. 118A-O another glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, optionally selected from -dibenzocyclooctyne, INX-SM-54-PAB-GlcA-dibenzocyclooctyne, INX-SM-1-PAB-GlcA-dibenzocyclooctyne, INX-SM-54-PAB-GlcA-dibenzocyclooctyne, INX-SM -33-PAB-GlcA-dibenzocyclooctyne, INX-SM-57-PAB-GlcA-dibenzocyclooctyne, INX-SM-7-PAB-GlcA-dibenzocyclooctyne, INX-SM-8-PAB-GlcA-dibenzo Cyclooctyne, INX-SM-2-PAB-GlcA-dibenzocyclooctyne, INX-SM-5-PAB-GlcA-dibenzocyclooctyne, INX-SM-6-PAB-GlcA-dibenzocyclooctyne, INX-SM-35 -PAB-GlcA-dibenzocyclooctyne, INX-SM-9-PAB-GlcA-dibenzocyclooctyne, INX-SM-10-PAB-GlcA-dibenzocyclooctyne, INX-SM-31-PAB-GlcA-dibenzocyclooctyne , INX-SM-32-PAB-GlcA-dibenzocyclooctyne, INX-SM-27-PAB-GlcA-dibenzocyclooctyne, INX-SM-35-PAB-GlcA-dibenzocyclooctyne, INX-SM-28-PAB -GlcA-dibenzocyclooctyne, INX-SM-34-PAB-GlcA-dibenzocyclooctyne, INX-SM-40-PAB-GlcA-dibenzocyclooctyne, INX-SM-3-PAB-GlcA-dibenzocyclooctyne, or GlcA and/or PAB is replaced with a different cleavable peptide or non-peptide linker, and another INX or INX-SM payload is included therein, optionally selected from those of FIGS. 118A-O. or (xviii) INX-SM-3-PAB-GlcA-NHS ester, INX-SM-53-PAB-GlcA-NHS ester, INX-SM-4-PAB-GlcA-NHS ester, INX-SM-56-PAB-GlcA-NHS ester, INX-SM-54-PAB-GlcA-NHS ester, INX-SM-8-PAB-GlcA -NHS ester, INX-SM-2-PAB-GlcA-NHS ester, INX-SM-7-PAB-GlcA-NHS ester, INX-SM-57-PAB-GlcA-NHS ester, INX-SM-32-PAB -GlcA-NHS ester, INX-SM-33-PAB-GlcA-NHS ester, INX-SM-31-PAB-GlcA-NHS ester, INX-SM-9-PAB-GlcA-NHS ester, INX-SM-10 -PAB-GlcA-NHS ester, INX-SM-35-PAB-GlcA-NHS ester, INX-SM-27-PAB-GlcA-NHS ester, INX-SM-28-PAB-GlcA-NHS ester, INX-SM -40-PAB-GlcA-NHS ester, INX-SM-34-PAB-GlcA-NHS ester, INX-SM-1-PAB-GlcA-NHS ester, or GlcA and/or PAB are different cleavable peptide linkers or a non-peptide linker, and another INX or INX-SM payload is substituted with the INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O. Corticoid agonist (payload) linker conjugate, or (xix) INX-SM-3-PAB-GlcA-maleimide, INX-SM-4-PAB-GlcA-maleimide, INX-SM-53-PAB-GlcA-maleimide, INX -SM-31-PAB-GlcA-maleimide, INX-SM-32-PAB-GlcA-maleimide, INX-SM-33-PAB-GlcA-maleimide, INX-SM-53-PAB-GlcA-maleimide, INX-SM -7-PAB-GlcA-maleimide, INX-SM-8-PAB-GlcA-maleimide, INX-SM-2-PAB-GlcA-maleimide, INX-SM-56-PAB-GlcA-maleimide, INX-SM-6 -PAB-GlcA-maleimide, INX-SM-54-PAB-GlcA-maleimide, INX-SM-1-PAB-GlcA-maleimide, INX-SM-9-PAB-GlcA-maleimide, INX-SM-35-PAB -GlcA-maleimide, INX-SM-27-PAB-GlcA-maleimide, INX-SM-28-PAB-GlcA-maleimide, INX-SM-34-PAB-GlcA-maleimide, INX-SM-40-PAB-GlcA 118A- another glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, optionally selected from those of GlcA-tetrazine, INX-SM-57-PAB-GlcA-tetrazine, INX-SM-7-PAB-GlcA-tetrazine, INX-SM-8-PAB-GlcA-tetrazine, INX-SM-2-PAB-GlcA- Tetrazine, INX-SM-56-PAB-GlcA-tetrazine, INX-SM-6-PAB-GlcA-tetrazine, INX-SM-54-PAB-GlcA-tetrazine, INX-SM-4-PAB-GlcA-tetrazine, INX-SM-9-PAB-GlcA-tetrazine, INX-SM-35-PAB-GlcA-tetrazine, INX-SM-27-PAB-GlcA-tetrazine, INX-SM-28-PAB-GlcA-tetrazine, INX- SM-34-PAB-GlcA-tetrazine, INX-SM-40-PAB-GlcA-tetrazine, INX-SM-10-PAB-GlcA-tetrazine, or GlcAy and/or PAB with different cleavable peptide linkers or non- Another glucocorticoid agonist is substituted with a peptide linker and another INX or INX-SM payload is substituted with the INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O. (payload) linker conjugate, or (xxi) INX-SM-1-PAB-GlcA-amine, INX-SM-3-PAB-GlcA-amine, INX-SM-53-PAB-GlcA-amine, INX-SM -6-PAB-GlcA-amine, INX-SM-54-PAB-GlcA-amine, INX-SM-8-PAB-GlcA-amine, INX-SM-2-PAB-GlcA-amine, INX-SM-56 -PAB-GlcA-amine, INX-SM-4-PAB-GlcA-amine, INX-SM-35-PAB-GlcA-amine, INX-SM-8-PAB-GlcA-amine, INX-SM-10-PAB -GlcA-amine, INX-SM-31-PAB-GlcA-amine, INX-SM-32-PAB-GlcA-amine, INX-SM-33-PAB-GlcA-amine, INX-SM-57-PAB-GlcA -Amine, INX-SM-27-PAB-GlcA-amine, INX-SM-35-PAB-GlcA-amine, INX-SM-34-PAB-GlcA-amine, INX-SM-28-PAB-GlcA-amine , INX-SM-40-PAB-GlcA-amine, INX-SM-7-PAB-GlcA-amine, or GlcA and/or PAB is replaced with a different cleavable peptide linker or non-peptide linker and another INX or another glucocorticoid agonist (payload) linker conjugate in which the INX-SM payload is replaced with the INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O, or ( xxii) Alkoxyamine-GlcA-PAB-DMEDA-INX-SM3, or Alkoxyamine-GlyGlu-PAB-DMEDA-INX-SM3, or the same, where the linker is attached to the same or different INX steroid via C11-OH or other linker payloads, including different peptide or non-peptide linkers,
(xxiii) Bromoacetyl-GlcA-PAB-DMEDA-INX-SM3, or Bromoacetyl--GlyGlu-PAB-DMEDA-INX-SM3, or a linker is attached to the same or different INX steroid via C11-OH , other linker payloads containing the same or different peptide or non-peptide linkers,
(xxiv) dibenzocyclooctyne-GlcA-PAB-DMEDA-INX-SM3, or dibenzocyclooctyne-GlyGlu-PAB-DMEDA-INX-SM3, or a linker attached to the same or different INX steroid via C11-OH; other linker payloads, including the same or different peptide or non-peptide linkers,
(xxv) Tetrazine-GlcA-PAB-DMEDA-INX-SM3, or Tetrazine-GlyGlu-PAB-DMEDA-INX-SM3, or the linker is attached to the same or different INX steroid via C11-OH, the same or other linker payloads, including different peptide or non-peptide linkers,
(xxvi) Alkoxyamine-GlcA-PAB-DMEDA-INX-SM3, or Alkoxyamine-GlyGlu-PAB-DMEDA-INX-SM3, or the linker is attached to the same or different INX steroid via C17, the same or other linker payloads, including different INX steroid payloads,
(xxvii) Bromoacetyl-GlcA-PAB-DMEDA-INX-SM3, or Bromoacetyl-GlyGlu-PAB-DMEDA-INX-SM3, or the linker is attached to the same or different INX steroid via C17, the same or other linker payloads, including different INX steroid payloads,
(xxviii) Maleimide-GlcA-PAB-DMEDA-INX-SM3, or Maleimide-GlyGlu-PAB-DMEDA-INX-SM3, or the same or different INX, where the linker is attached to the same or different INX steroid via C17; other linker payloads, including steroid payloads,
(xxix) dibenzocyclooctyne-GlcA-PAB-DMEDA-INX-SM3, or dibenzocyclooctyne-GlyGlu-PAB-DMEDA-INX-SM3, or the linker is attached to the same or different INX steroid via C17; other linker payloads, including the same or different INX steroid payloads;
(xxx) Tetrazine-GlcA-PAB-DMEDA-INX-SM3, or Tetrazine-GlyGlu-PAB-DMEDA-INX-SM3, or the same or different INX where the linker is attached to the same or different INX steroid via C17 Other linker payloads, including steroid payloads, and (xxxi) Amine-GlcA-PAB-DMEDA-INX-SM3, or Amine-GlyGlu-PAB-DMEDA-INX-SM3, or INXs with the same or different linkers via C17. It is a specific object of the present invention to provide a glucocorticoid agonist (payload) linker conjugate selected from other linker payloads, including the same or a different linker, attached to a steroid payload.

13. An antibody drug conjugate (ADC) selected from:
(i) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (alkoxyamine + ketone conjugation (C11-OH bond), or INX-SM payload Another ADC containing a different INX-SM payload, conjugated to an antibody via alkoxyamine + ketone conjugation and C11-OH linked,
(ii) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (azido + dibenzocyclooctyne conjugation (C11-OH bond), or INX-SM payload Another ADC containing a different INX-SM payload, conjugated to the antibody via azide + dibenzocyclooctyne conjugation and C11-OH linked,
(iii) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (haloacetyl + cysteine conjugation (C11-OH bond), or INX-SM payload is an azide + another ADC containing a different INX-SM payload, conjugated to the antibody via dibenzocyclooctyne conjugation and C11-OH linked;
(iv) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (maleimide + cysteine conjugation (C11-OH bond), or INX-SM payload is an azide + another ADC containing a different INX-SM payload, conjugated to the antibody via dibenzocyclooctyne conjugation and C11-OH linked;
(v) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (tetrazine + trans cyclooctene conjugation (C11-OH bond)), or INX-SM Another ADC containing a different INX-SM payload, where the payload is conjugated to an antibody via tetrazine + transcycloocte conjugation and is C11-OH linked;
(vi) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (alkoxyamine + ketone conjugation (C11-OH bond)), or INX-SM payload Another ADC containing a different INX-SM payload, which is conjugated to the antibody via alkoxyamine + ketone conjugation and is C11-OH linked,
(vii) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (azido + dibenzocyclooctyne conjugation (C17 bond)), or INX-SM payload Another ADC containing a different INX-SM payload, conjugated to an antibody via azide + dibenzocyclooctyne conjugation and C17-linked;
(viii) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (haloacetyl + cysteine conjugation (C17 bond)), or INX-SM payload is azide + Another ADC containing a different INX-SM payload, conjugated to an antibody via dibenzocyclooctyne conjugation and C17 linked,
(ix) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (tetrazine + trans cyclooctene conjugation (C17 bond)), or INX-SM linker payload Another ADC containing a different INX-SM payload, which is conjugated to the antibody via tetrazine + trans cyclooctene conjugation and C17-linked;
(x) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (amine+glutamine conjugation (C17 linkage) using transglutaminase), or INX- Another ADC containing a different INX-SM payload, where the SM linker payload is conjugated to the antibody via amine+glutamine conjugation using transglutaminase and C17-linked;
(xi) INX-SM-3-PAB-GlcA-Ab or INX-SM-3-PAB-Glu-Gly-Ab (alkoxyamine and ketone conjugation) (N-linked payload), or INX-SM linker payload is an alkoxy Another ADC containing a different INX-SM payload, conjugated and N-linked to the antibody via amine and ketone conjugation,
(xii) INX-SM-3-PAB-GlcA-Ab or INX-SM-3-PAB-Glu-Gly-Ab (haloacetyl conjugation) (N-linked payload), or INX-SM linker payload is a haloacetyl conjugation another ADC containing a different INX-SM payload, conjugated to the antibody via conjugation and N-linked;
(xiii) INX-SM-3-PAB-GlcA-Ab or INX-SM-3-PAB-Glu-Gly-Ab (azide + dibenzocyclooctyne conjugation) (N-linked payload), or INX-SM linker payload Another ADC containing a different INX-SM payload, conjugated and N-linked to the antibody via azide + dibenzocyclooctyne conjugation,
(xiv) INX-SM-3-GlcA-Ab or INX-SM-3-Glu-Gly-Ab (N-hydroxysuccinimide conjugation) (N-linked payload), or INX-SM linker payload is a N-hydroxysuccinimide conjugate; another ADC containing a different INX-SM payload, conjugated to the antibody via conjugation and N-linked;
(xv) INX-SM-3-PAB-GlcA-Ab or INX-SM-3-PAB-Glu-Gly-Ab (azide + dibenzocyclooctyne conjugation) (N-linked payload), or INX-SM linker payload Another ADC containing a different INX-SM payload, conjugated and N-linked to the antibody via azide + dibenzocyclooctyne conjugation,
(xvi) INX-SM-3-PAB-GlcA-Ab or INX-SM-3-PAB-Glu-Gly-Ab (N-hydroxysuccinimide conjugation) (N-linked payload), or INX-SM linker payload is N - another ADC containing a different INX-SM payload, conjugated and N-linked to the antibody via hydroxysuccinimide conjugation,
(xvii) INX-SM-3-Glu-Gly-Ab or INX-SM-3-PAB-Glu-Gly-Ab (maleimide conjugation) (N-linked payload), or INX-SM linker payload conjugates maleimide another ADC containing a different INX-SM payload, conjugated to an antibody via
(xviii) INX-SM-3-Glu-Gly-Ab or INX-SM-3-PAB-Glu-Gly-Ab or INX-SM-3-PAB-GlcA-Ab (trans cyclooctene + tetrazine conjugation) (N-linked payload), or another ADC containing a different INX-SM payload, where the INX-SM linker payload is conjugated to the antibody via transcyclooctene+tetrazine conjugation and is N-linked;
(xix) INX-SM-3-Glu-Gly-Ab or INX-SM-3-PAB-Glu-Gly-Ab or INX-SM-3-PAB-GlcA-Ab (amine conjugation) (N-linked payload) , or another ADC containing a different INX-SM payload, in which the INX-SM linker payload is conjugated and N-linked to the antibody via transcyclooctene+tetrazine conjugation. Gate (ADC).

式中、
Ａｂ＝抗体、好ましくは、ヒト免疫細胞に結合する抗体、好ましくは、生理学的ｐＨにおいてヒトＶＩＳＴＡ免疫細胞に結合する抗ＶＩＳＴＡ抗体、
Ｌ＝リンカー、
ＡＡ＝単一、二重、または三重アミノ酸配列、
ＮＨペイロード＝

ＲＥＧが、独立して、水素、アルキル、ビフェニル、－ＣＦ３、－ＮＯ２、－ＣＮ、フルオロ、ブロモ、クロロ、アルコキシル、アルキルアミノ、ジアルキルアミノ、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－からなる群から選択され、

Ａｂ＝抗体、
Ｌ＝リンカー、
ＡＡ＝単一、二重、または三重アミノ酸配列、
ＮＨペイロード＝

ＲＥＧが、独立して、水素、アルキル、ビフェニル、－ＣＦ３、－ＮＯ２、－ＣＮ、フルオロ、ブロモ、クロロ、アルコキシル、アルキルアミノ、ジアルキルアミノ、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－からなる群から選択され、

Ａｂ＝抗体、
Ｌ＝リンカー、
ＡＡ＝単一、二重、もしくは三重アミノ酸配列、または存在しない、
ＮＨペイロード＝

ＲＥＧが、独立して、水素、アルキル、ビフェニル、－ＣＦ３、－ＮＯ２、－ＣＮ、フルオロ、ブロモ、クロロ、アルコキシル、アルキルアミノ、ジアルキルアミノ、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－からなる群から選択され、
ＲＴ＝ＡＡまたは

Ａｂ＝抗体、任意選択的に、抗ヒトＶＩＳＴＡ抗体、
Ｌ＝リンカー、
ＡＡ＝単一、二重、または三重アミノ酸配列、
Ｏペイロード＝

Ａｂ＝抗体、典型的には、免疫細胞、典型的には、ヒト免疫細胞、例えば、ＶＩＳＴＡ上に発現される抗原に結合する抗体、
Ｌ＝リンカー、
ＡＡ＝単一、二重、もしくは三重アミノ酸配列、または存在しない、
Ｏペイロード＝

ＲＴ＝ＡＡまたは

である、抗体薬物コンジュゲート（ＡＤＣ）を提供することが、本発明の別の具体的な目的である。 An antibody drug conjugate (ADC) selected from:

During the ceremony,
Ab = antibody, preferably an antibody that binds to human immune cells, preferably an anti-VISTA antibody that binds to human VISTA immune cells at physiological pH;
L = linker,
AA = single, double, or triple amino acid sequence;
NH payload =

REG is independently hydrogen, alkyl, biphenyl, -CF3, -NO2, -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino-C( O)-, and dialkylamino C(O)-,

Ab=antibody,
L = linker,
AA = single, double, or triple amino acid sequence;
NH payload =

REG is independently hydrogen, alkyl, biphenyl, -CF3, -NO2, -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino-C( O)-, and dialkylamino C(O)-,

Ab=antibody,
L = linker,
AA = single, double or triple amino acid sequence or absent;
NH payload =

REG is independently hydrogen, alkyl, biphenyl, -CF3, -NO2, -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino-C( O)-, and dialkylamino C(O)-,
RT=AA or

Ab = antibody, optionally anti-human VISTA antibody,
L = linker,
AA = single, double, or triple amino acid sequence;
O payload =

Ab = antibody, typically an antibody that binds to an antigen expressed on an immune cell, typically a human immune cell, such as VISTA;
L = linker,
AA = single, double or triple amino acid sequence or absent;
O payload =

RT=AA or

It is another specific object of the present invention to provide antibody drug conjugates (ADCs) that are.

It is a specific object of the present invention to provide an antibody drug conjugate (ADC) as described above, wherein the linker comprises a cleavable or non-cleavable peptide or a sacrificial linker.

PAB and/or amino acids or peptides, optionally 1 to 12 amino acids, more optionally dipeptides, tripeptides, quatrapeptides, pentapeptides, more optionally Gly, Asn, Asp, Gln, Leu, Lys, Ala, Phe, Cit, Val, Val-Cit, Val-Ala, Val-Gly, Val-Gln, Ala-Val, Cit-Cit, Lys-Val-Cit, Asp-Val-Ala, Ala-Ala-Asn, Asp-Val-Ala, Ala-Val-Cit, Ala-Asn-Val, βAla-Leu-Ala-Leu, Lys-Val-Ala, Val-Leu-Lys, Asp-Val-Cit, It is a specific object of the present invention to provide an antibody drug conjugate (ADC) according to any of the foregoing, comprising a linker selected from Val-Ala-Val, and Ala-Ala-Asn.

から選択され、式中、ｎ＝２～１２、２～１０、２～８、２～６、２～４であり、Ａが、抗体またはその抗原結合断片、好ましくは、免疫細胞、好ましくは、ヒト免疫細胞上に発現される抗原に結合する抗体または抗体断片、例示的実施形態では、抗ヒトＶＩＳＴＡ抗体である、ステロイド抗体コンジュゲートを提供することが、本発明の具体的な目的である。 Structure below:

where n=2-12, 2-10, 2-8, 2-6, 2-4, and A is an antibody or antigen-binding fragment thereof, preferably an immune cell, preferably It is a specific object of the invention to provide steroid antibody conjugates that bind to antigens expressed on human immune cells, in exemplary embodiments anti-human VISTA antibodies.

式中、
Ｘが、フェニル、スピロ［３．３］ヘプタン、３～６員複素環、シクロアルキル、スピロアルキル、スピロヘテロシクロアルキル、二環式アルキル、ヘテロ二環式アルキル、［１．１．１］ビシクロペンタン、ビシクロ［２．２．２］オクタン、アダマンタン、及びキュバンから選択され、これらの各々を、独立して、Ｆ、Ｃｌ、Ｂｒ、Ｉ、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換することができ、その環構造の各々が、Ｎ、Ｓ、及びＯから選択される少なくとも１つの骨格ヘテロ原子を含み得、任意選択的に、１～４個のＣ１～３アルキルまたはＣ１～３ペルフルオロアルキルで更に置換され、
Ｚが、フェニル、スピロ［３．３］ヘプタン、３～６員複素環、シクロアルキル、スピロアルキル、スピロヘテロシクロアルキル、二環式アルキル、ヘテロ二環式アルキル、［１．１．１］ビシクロペンタン、ビシクロ［２．２．２］オクタン、アダマンタン、及びキュバンから選択され、これらの各々を、独立して、Ｆ、Ｃｌ、Ｂｒ、Ｉ、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換することができ、その環構造の各々が、Ｎ、Ｓ、及びＯから選択される少なくとも１つの骨格ヘテロ原子を含み得、任意選択的に、１～４個のＣ１～３アルキルまたはＣ１～３ペルフルオロアルキルで更に置換され、
Ｙが、ＣＨＲ１、Ｏ、Ｓ、及びＮＲ１から選択され、
Ｅが、ＣＨ２及びＯから選択され、
Ｇが、ＣＨ及びＮから選択され、
更に、ＧがＣＨであり、Ｘがフェニルであるときに、Ｚがフェニルではなく、
ＸへのＧの結合が、任意選択的にＣ１～３アルキル及びエチレンオキシドから選択され得、これらの各々が、独立して、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換され得、任意選択的に、１～４個のＣ１～３アルキルで更に置換され、
ＺへのＸの結合が、Ｘ及びＺ上の任意の利用可能な位置を占有し得、
置換基ＮＲ１Ｒ２が、Ｚ上の任意の利用可能な位置を占有し得、
Ｒ１が、Ｈ、１～８個の炭素の直鎖または分岐アルキル、アリール、及びヘテロアリール基から選択され、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｒ１がＨであるとき、Ｒ２が、Ｈ、１～８個の炭素の直鎖または分岐アルキル、アリール、及びヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｒ１がＨ、１～８個の炭素の直鎖もしくは分岐アルキル、またはヘテロアリールであるとき、Ｒ２が、
［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］ｍ－［Ｃ＝Ｏ］－［Ｖ］ｋ－Ｊ、
Ｃ＝Ｏ）ＯＣＨ２－ｐ－アミノフェニル－Ｎ－Ｖ－Ｊ、
（Ｃ＝Ｏ）ＯＣＨ２－ｐ－アミノフェニル－Ｎ－［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］ｍ－［Ｃ＝Ｏ］－［Ｖ］ｋ－Ｊ、及び
［Ｖ］ｋ－（Ｃ＝Ｏ）ＯＣＨ２－ｐ－アミノフェニル－Ｎ－［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］ｍ－［Ｃ＝Ｏ］－Ｊ、から選択される官能基であり得、
式中、ｍ＝１～６であり、ｋ＝０～１であり、Ｗの各順列が、独立して、Ｈ、ｎ＝１～４である［（ＣＨ２）ｎＲ３］、Ｒ３で終端する分岐アルキル鎖、及び１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基から選択され得、
Ｒ３が、Ｈ、メチル、エチル、イソプロピル、ＯＨ、Ｏ－アルキル、ＮＨ２、ＮＨ－アルキル、Ｎ－ジアルキル、ＳＨ、Ｓ－アルキル、グアニジン、尿素、カルボン酸、カルボキサミド、カルボン酸エステル、置換または非置換アリール、置換または非置換ヘテロアリールから選択され、当該アリール及びヘテロアリール置換基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択され得、
Ｖが、１～８個の炭素のアルキル鎖、１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基、１～８個の炭素を含む直鎖または分岐アルキル基、－Ｏ－アルキル、カルボン酸、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－、１～３アミノ酸配列であって、各アミノ酸が、独立して、Ｇｌｕ、Ｇｌｙ、Ａｓｎ、Ａｓｐ、Ｇｌｎ、Ｌｅｕ、Ｌｙｓ、Ａｌａ、βＡｌａ、Ｐｈｅ、Ｖａｌ、及びＣｉｔから選択される、１～３アミノ酸配列、アリール、ならびにヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｊが、－ＮＨ２、Ｎ３、チオ、シクロオクチン、－ＯＨ、－ＣＯ２Ｈ、トランスシクロオクテン、アルキニル、プロパルギル、

から選択される反応基であり、
式中、Ｒ３２が、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ３３が、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ３４が、Ｈ、Ｍｅ、テトラジン－Ｈ、及びテトラジン－Ｍｅであり、
Ｒ５が、－ＣＨ２ＯＨ、－ＣＨ２ＳＨ、－ＣＨ２Ｃｌ、－ＳＣＨ２Ｃｌ、－ＳＣＨ２Ｆ、－ＳＣＨ２ＣＦ３、ヒドロキシ、－ＯＣＨ２ＣＮ、－ＯＣＨ２Ｃｌ、－ＯＣＨ２Ｆ、－ＯＣＨ３、－ＯＣＨ２ＣＨ３、－ＳＣＨ２ＣＮ、及び

からなる群から選択され、
Ｒ６及びＲ７が、独立して、水素及びＣ１～１０アルキルから選択され、
Ｑが、Ｈ、

Ｒ８が１～８個の炭素の直鎖もしくは分岐アルキルである、Ｃ（Ｏ）Ｒ８、またはｎ＝１～４であり、Ｒ４＝Ｈ、アルキルもしくは分岐アルキル、またはＰ（Ｏ）ＯＲ４である、（Ｃ＝Ｏ）ＮＲ４ＣＨｎＮＲ４（Ｃ＝Ｏ）ＯＣＨ２－（Ｖ）ｎ－Ｊであり得、
Ａ１及びＡ２が、独立して、Ｈ及びＦから選択され、
別途明記しない限り、全ての可能な立体異性体が特許請求される、グルココルチコイドアゴニスト化合物を提供することが、本発明の具体的な目的である。 A glucocorticoid agonist compound of formula (I),

During the ceremony,
X is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C1 to further substituted with 3 alkyl or C1-3 perfluoroalkyl,
Z is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C1 to further substituted with 3 alkyl or C1-3 perfluoroalkyl,
Y is selected from CHR1, O, S, and NR1;
E is selected from CH2 and O;
G is selected from CH and N;
Furthermore, when G is CH and X is phenyl, Z is not phenyl,
The bond of G to optionally further substituted with 1 to 4 C1-3 alkyl,
the bond of X to Z may occupy any available position on X and Z;
The substituent NR1R2 may occupy any available position on Z;
R1 is selected from H, linear or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and the aryl and heteroaryl groups are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH , -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O) -, and dialkylamino C(O)-,
When R1 is H, R2 may be selected from H, straight chain or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and the aryl and heteroaryl groups are alkyl, haloalkyl, halogen, Biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O- , alkylamino-C(O)-, and dialkylamino-C(O)-,
When R1 is H, linear or branched alkyl of 1 to 8 carbons, or heteroaryl, R2 is
[(C=O)CH(W)NH]m-[C=O]-[V]kJ,
C=O)OCH2-p-aminophenyl-NVJ,
(C=O)OCH2-p-aminophenyl-N-[(C=O)CH(W)NH]m-[C=O]-[V]k-J, and [V]k-(C= O) OCH2-p-aminophenyl-N-[(C=O)CH(W)NH]m-[C=O]-J,
where m=1 to 6, k=0 to 1, and each permutation of W is independently H, n=1 to 4 [(CH2)nR3], a branch terminating in R3 may be selected from alkyl chains, and linear or branched polyethylene oxide groups containing from 1 to 13 units;
R3 is H, methyl, ethyl, isopropyl, OH, O-alkyl, NH2, NH-alkyl, N-dialkyl, SH, S-alkyl, guanidine, urea, carboxylic acid, carboxamide, carboxylic acid ester, substituted or unsubstituted selected from aryl, substituted or unsubstituted heteroaryl, wherein the aryl and heteroaryl substituents are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, may be selected from thiols, thioalkyls, guanidines, ureas, carboxylic acids, alkoxyls, carboxamides, carboxylic esters, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein the aryl and The heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl- may be substituted with a functional group selected from C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH2, N3, thio, cyclooctyne, -OH, -CO2H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R32 is selected from Cl, Br, F, mesylate, and tosylate, and R33 is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-(4- nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, R34 is H, Me, tetrazine-H, and tetrazine-Me;
R5 is -CH2OH, -CH2SH, -CH2Cl, -SCH2Cl, -SCH2F, -SCH2CF3, hydroxy, -OCH2CN, -OCH2Cl, -OCH2F, -OCH3, -OCH2CH3, -SCH2CN, and

selected from the group consisting of
R6 and R7 are independently selected from hydrogen and C1-10 alkyl;
Q is H,

R8 is straight chain or branched alkyl of 1 to 8 carbons, C(O)R8, or n=1 to 4 and R4=H, alkyl or branched alkyl, or P(O)OR4; (C=O)NR4CHnNR4(C=O)OCH2-(V)n-J;
A1 and A2 are independently selected from H and F;
It is a specific object of the present invention to provide glucocorticoid agonist compounds in which all possible stereoisomers are claimed, unless otherwise specified.

から選択され、これらの各々が、独立して、Ｆ、Ｃｌ、Ｂｒ、Ｉ、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換され得、任意選択的に、１～４個のＣ１～３アルキルまたはＣ１～３ペルフルオロアルキル基で更に置換され、
その環構造の各々が、Ｎ、Ｓ、及びＯから選択される少なくとも１つの追加の骨格ヘテロ原子を含み得、
式中、各

が、式の残りの部分への付着点を示し、当該付着点の各々が、Ｎ、Ｓ、及びＯから選択される追加のヘテロ原子を介して式の残りの部分に共有結合され得る、前述のいずれかに記載のグルココルチコイドアゴニスト化合物を提供することが、本発明の具体的な目的である。 Z is

each of which may be independently substituted with 1 to 4 heteroatoms selected from F, Cl, Br, I, N, S, and O, optionally 1 to 4. further substituted with 4 C1-3 alkyl or C1-3 perfluoroalkyl groups,
each of the ring structures may include at least one additional skeletal heteroatom selected from N, S, and O;
During the ceremony, each

indicates a point of attachment to the remainder of the formula, each of which may be covalently bonded to the remainder of the formula through an additional heteroatom selected from N, S, and O. It is a specific object of the present invention to provide glucocorticoid agonist compounds according to any of the above.

から選択され、
これらの各々が、独立して、Ｆ、Ｃｌ、Ｂｒ、Ｉ、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換され得、任意選択的に、１～４個のＣ１～３アルキルまたはＣ１～３ペルフルオロアルキル基で更に置換され、
その環構造の各々が、Ｎ、Ｓ、及びＯから選択される少なくとも１つの追加の骨格ヘテロ原子を含み得、
式中、各

が、式の残りの部分への付着点を示し、当該付着点の各々が、Ｎ、Ｓ、及びＯから選択される追加のヘテロ原子を介して式の残りの部分に共有結合され得る、前述のいずれかに記載のグルココルチコイドアゴニスト化合物を提供することが、本発明の具体的な目的である。 Z-NR1 is

selected from
Each of these may be independently substituted with 1 to 4 heteroatoms selected from F, Cl, Br, I, N, S, and O, optionally with 1 to 4 C1 further substituted with ~3 alkyl or C1-3 perfluoroalkyl group,
each of the ring structures may include at least one additional skeletal heteroatom selected from N, S, and O;
During the ceremony, each

indicates a point of attachment to the remainder of the formula, each of which may be covalently bonded to the remainder of the formula through an additional heteroatom selected from N, S, and O. It is a specific object of the present invention to provide glucocorticoid agonist compounds according to any of the above.

式中、
Ｙが、ＣＨ_２及びＯから選択され、
Ｅが、ＣＨ_２及びＯから選択され、
Ｇが、ＣＨ及びＮから選択され、
Ｌが、Ｈ及びＦから選択され、
Ｒ_５が、－ＣＨ_２ＯＨ、－ＳＣＨ_２Ｆ、及び

から選択され、
Ａ_１及びＡ_２が、独立して、Ｈ及びＦから選択され、
Ｖが、１～８個の炭素のアルキル鎖、１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基、１～８個の炭素を含む直鎖または分岐アルキル基、－Ｏ－アルキル、カルボン酸、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－、１～３アミノ酸配列であって、各アミノ酸が、独立して、Ｇｌｕ、Ｇｌｙ、Ａｓｎ、Ａｓｐ、Ｇｌｎ、Ｌｅｕ、Ｌｙｓ、Ａｌａ、βＡｌａ、Ｐｈｅ、Ｖａｌ、及びＣｉｔから選択される、１～３アミノ酸配列、アリール、ならびにヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｊが、－ＮＨ２、Ｎ３、チオ、シクロオクチン、－ＯＨ、－ＣＯ２Ｈ、トランスシクロオクテン、アルキニル、プロパルギル、

から選択される反応基であり、
式中、Ｒ３２が、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ３３が、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ３４が、Ｈ、Ｍｅ、テトラジン－Ｈ、及びテトラジン－Ｍｅである、グルココルチコイドアゴニスト化合物を提供することが、本発明の具体的な目的である。 A glucocorticoid agonist compound having the structure of formula (II),

During the ceremony,
Y is selected from CH ₂ and O;
E is selected from CH ₂ and O;
G is selected from CH and N;
L is selected from H and F;
R ₅ is -CH ₂ OH, -SCH ₂ F, and

selected from
A ₁ and A ₂ are independently selected from H and F;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein the aryl and The heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl- may be substituted with a functional group selected from C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH2, N3, thio, cyclooctyne, -OH, -CO2H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R32 is selected from Cl, Br, F, mesylate, and tosylate, and R33 is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-(4- nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R34 is H, Me, tetrazine-H, and tetrazine-Me, This is a specific object of the present invention.

式中、
Ｙが、ＣＨ２及びＯから選択され、
Ｅが、ＣＨ２及びＯから選択され、
Ｇが、ＣＨ及びＮから選択され、
Ｌが、Ｈ及びＦから選択され、
Ｒ５が、－ＣＨ２ＯＨ、－ＳＣＨ２Ｆ、及び

から選択され、
Ａ１及びＡ２が、独立して、Ｈ及びＦから選択され、
Ｖが、１～８個の炭素のアルキル鎖、１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基、１～８個の炭素を含む直鎖または分岐アルキル基、－Ｏ－アルキル、カルボン酸、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－、１～３アミノ酸配列であって、各アミノ酸が、独立して、Ｇｌｕ、Ｇｌｙ、Ａｓｎ、Ａｓｐ、Ｇｌｎ、Ｌｅｕ、Ｌｙｓ、Ａｌａ、βＡｌａ、Ｐｈｅ、Ｖａｌ、及びＣｉｔから選択される、１～３アミノ酸配列、アリール、ならびにヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｊが、－ＮＨ２、Ｎ３、チオ、シクロオクチン、－ＯＨ、－ＣＯ２Ｈ、トランスシクロオクテン、アルキニル、プロパルギル、

から選択される反応基であり、
式中、Ｒ３２が、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ３３が、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ３４が、Ｈ、Ｍｅ、テトラジン－Ｈ、及びテトラジン－Ｍｅである、前述のいずれかに記載のグルココルチコイドアゴニスト化合物を提供することが、本発明の具体的な目的である。
前述のいずれかに記載のグルココルチコイドアゴニスト化合物であって、

から選択される、グルココルチコイドアゴニスト化合物を提供することが、本発明の具体的な目的である。 has the structure of formula (III),

During the ceremony,
Y is selected from CH2 and O;
E is selected from CH2 and O;
G is selected from CH and N;
L is selected from H and F;
R5 is -CH2OH, -SCH2F, and

selected from
A1 and A2 are independently selected from H and F;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein the aryl and The heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl- may be substituted with a functional group selected from C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH2, N3, thio, cyclooctyne, -OH, -CO2H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R32 is selected from Cl, Br, F, mesylate, and tosylate, and R33 is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-(4- the glucocorticoid agonist according to any of the preceding, wherein R34 is H, Me, tetrazine-H, and tetrazine-Me; It is a specific object of the present invention to provide compounds.
A glucocorticoid agonist compound according to any of the foregoing,

It is a specific object of the present invention to provide glucocorticoid agonist compounds selected from:

as described in any of the foregoing i.e. formulas I, II, wherein X or Z may be spiro[3.3]heptane or [1.1.1]bicyclopentane and Y may be CH2 or O It is a specific object of the present invention to provide glucocorticoid agonist compounds of , or III.

An antibody or antibody-binding fragment thereof, preferably one that binds to an antigen expressed by an immune cell, preferably a human immune cell, wherein the antibody or antibody-binding fragment is as described in any one of the previous embodiments. It is a specific object of the present invention to provide an antibody drug conjugate (ADC) attached to at least one glucocorticoid agonist.

から選択され、好ましくは、式中、ｎ＝２～１２、２～１０、２～８、２～６、または２～４であり、Ａが、免疫細胞、好ましくは、ヒト免疫細胞よって発現される抗原に結合する抗体、いくつかの例示的実施形態では、抗ヒトＶＩＳＴＡ抗体である、ＡＤＣを提供することが、本発明の具体的な目的である。 An ADC,

preferably, where n=2-12, 2-10, 2-8, 2-6, or 2-4, and A is expressed by an immune cell, preferably a human immune cell. It is a specific object of the invention to provide an ADC that binds to an antigen, in some exemplary embodiments, an anti-human VISTA antibody.

A composition comprising at least one glucocorticoid agonist compound as described above, or a steroid linker conjugate of Formula I, II, or III, or an ADC containing it, and a pharmaceutically acceptable carrier. It is a specific object of the present invention to provide.

It is a specific object of the present invention to provide compositions as described above that are suitable for in vivo administration to a subject in need thereof.

It is a particular object of the invention to provide a composition as described above, comprising at least one additive.

It is a particular object of the invention to provide a composition as described above, comprising at least one stabilizer or buffer.

It is a particular object of the present invention to provide a composition as described above, which is suitable for parenteral administration, optionally by injection.

Optionally, providing a composition as described above is suitable for injection into a subject in need thereof via intravenous, subcutaneous, intramuscular, intratumoral, or intrathecal injection. , is a specific object of the present invention.

It is a specific object of the present invention to provide a composition as described above, which can be administered subcutaneously, intramuscularly or intravenously.

A device providing for subcutaneous administration selected from the group consisting of a syringe, an injection device, an infusion pump, an injector pen, a needleless device, an autoinjector, and a subcutaneous patch delivery system, optionally administering to the patient a glucocorticoid receptor agonist or It is a specific object of the present invention to provide a composition as described above included in a device for delivering a fixed dose of an ADC containing it.

a glucocorticoid agonist compound or steroid-linker conjugate or ADC as described in any of the foregoing, or for the treatment, prophylaxis, or inhibition of inflammation, allergy, or autoimmunity in a subject in need thereof; It is a specific object of the present invention to provide the use of a composition containing.

A glucocorticoid agonist compound according to any of the foregoing, or a steroid linker conjugate of Formula I, II, or III, or an ADC containing it, or in need of the treatment, prevention, or inhibition of inflammatory or autoimmune or allergic reactions. It is a specific object of the present invention to provide compositions containing for use in the preparation of medicaments for treatment, prophylaxis, or inhibition in a subject.

ADC containing at least one glucocorticoid agonist compound as described in any of the foregoing, or a steroid linker conjugate of Formula I, II, or III, or as described in any of the foregoing, for treatment and It is a specific object of the present invention to provide a method of treatment and/or prophylaxis comprising administering to a subject in need of such prophylaxis.

In human subjects, for the treatment of allergy, autoimmunity, transplantation, gene therapy, inflammation, GVHD, or sepsis, or to treat inflammatory, autoimmune, or allergic side effects associated with any of the aforementioned conditions. It is a specific object of the present invention to provide a use, medicament, composition or method as described in any of the foregoing, for or for the prevention.

It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, which is for acute use.

It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, which is for chronic use.

It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, which is for maintenance therapy.

For the treatment or prevention of acute or chronic inflammation and autoimmune and inflammatory indications associated therewith, optionally where the condition is, inter alia, acquired aplastic anemia+, acquired hemophilia+ , acute disseminated encephalomyelitis (ADEM)+, acute hemorrhagic leukoencephalitis (AHLE)/Hurst disease+, primary agammaglobulinemia+, alopecia areata+, ankylosing spondylitis (AS), anti-NMDA receptor somatic encephalitis+, antiphospholipid syndrome (APS)+, arteriosclerosis, autism spectrum disorder (ASD), autoimmune Addison's disease (AAD)+, autoimmune autonomic neuropathy/autoimmune autonomic ganglionopathy (AAG), autoimmune encephalitis+, autoimmune gastritis, autoimmune hemolytic anemia (AIHA)+, autoimmune hepatitis (AIH)+, autoimmune hyperlipidemia, autoimmune hypophysitis/lymph Globular hypophysitis+, autoimmune inner ear disease (AIED)+, autoimmune lymphoproliferative syndrome (ALPS)+, autoimmune myocarditis, autoimmune oophoritis+, autoimmune orchitis+, autoimmunity pancreatitis (AIP)/immunoglobulin G4-related disease (IgG4-RD)+, type I, type II, and type III autoimmune polyglandular syndrome+, autoimmune progesterone dermatitis+, autoimmune sudden deafness ( SNHL), atonia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, Autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis , autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy (AMAN), Barro's disease, Behcet's disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease , Chagas disease, chronic inflammatory demyelinating polyneuritis (CIDP), chronic relapsing polyosteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial syndrome Pemphigus, Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, type 1 diabetes, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler syndrome , endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrotic alveoli inflammation, fibrosing alveolitis, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schön Rhein purpura (HSP), herpes gestationalis or pemphigoid (PG), hidraden abscess (HS) (opposite acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune Thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert's disease. Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (including nephritis and skin), chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucka-Habelmann disease, multiple motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasia gravis, myelin oligodendrocyte glycoprotein antibodies disorders, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, opsoclonus-myoclonus syndrome (OMS), rheumatoid arthritis (PR), PANDAS, paraneoplastic Cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, Pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, type I, II, III polyglandular syndrome, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary Biliary cholangitis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, erythroblastic aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiffness Person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura ( TTP), thyroid eye disease (TED), Tolosa Hunt syndrome (THS), transverse myelitis, type 1 diabetes, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease. It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising:

For the treatment or prevention of acute or chronic inflammation and its associated autoimmune and inflammatory and allergic indications or side effects, the condition being optionally severe asthma, giant cell arteritis, ANKA vasculitis. It is a specific object of the present invention to provide a use, medicament, composition or method according to any of the foregoing, including colitis and IBD (colitis and Crohn's disease).

Rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, sweat gland abscess, uveitis, Behcet's disease, spondyloarthropathy, or It is a particular object of the invention to provide a use, medicament, composition or method as described in any of the above for the treatment or prevention of conditions selected from psoriasis.

One or more of the following:
(i) chronic, acute, episodic allergies, inflammation, or inflammatory conditions, such as chronic, acute, episodic, and/or remitting/relapsing;
(ii) a condition that can be effectively treated only primarily with high doses of steroids; optionally, the patient is or is being treated with high doses of steroids; asthma; , COPD, polymyalgia rheumatica and/or giant cell arteritis,
(iii) Conditions with comorbidities that limit steroid use, optionally diabetes, nonalcoholic steatohepatitis (NASH), morbid obesity, avascular necrosis/osteonecrosis (AVN), glaucoma, steroid-induced high blood pressure, severe skin fragility, and/or osteoarthritis;
(iv) conditions for which safe long-term therapeutic agents are available, but where induction for several months with high doses of steroids is desired; optionally, induction for several months with high doses of steroids is therapeutic; AAV, polymyositis, dermatomyositis, lupus, inflammatory lung disease, autoimmune hepatitis, inflammatory bowel disease, immune thrombocytopenia, autoimmune hemolytic anemia, gout patients,
(v) skin conditions requiring short-term/long-term treatment, optionally conditions of uncertain treatment or duration, and/or conditions for which there are no effective alternatives to steroid administration, optionally Stevens Johnson, other severe drug eruption conditions, conditions with extensive contact dermatitis, other severe immune-related skin conditions such as PG, LCV, erythroderma, and the like;
(vi) conditions treated with high-dose corticosteroids for flare-ups/relapses, optionally COPD, asthma, lupus, gout, pseudogout;
(vii) immune-related neurological diseases such as small fiber neuropathy, MS (subset), chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, and the like;
(viii) optionally hematology/oncology indications for which high doses of steroids are therapeutically warranted or beneficial;
(ix) ophthalmological conditions, optionally uveitis, iritis, scleritis, and the like;
(x) conditions associated with persistent or very long-term adrenal insufficiency or secondary adrenal insufficiency, optionally an iatrogenic Addison's disease crisis;
(xi) treatment or prophylaxis in patients with conditions often treated with long-term, low-dose steroids, optionally lupus, RA, psA, vasculitis, or (xii) any combination of any of the foregoing. It is a specific object of the present invention to provide a use, medicament, composition or method according to any of the foregoing for.

For treatment or prophylaxis in special classes of patients at risk of toxicity on steroid therapy, such as pregnant/lactating women, pediatric patients, and optionally pediatric patients with growth defects or cataracts. It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, wherein the patient is further treated with another active agent.

The patient has received an immunoinhibitory antibody or fusion protein that targets one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA, and and/or further treated with an immunomodulatory antibody or fusion protein selected from agonistic antibodies or fusion proteins targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28, or ICOS. It is a specific object of the invention to provide any use, medicament, composition or method.

the ADC comprises, and the ADC requires, an antibody or antigen-binding fragment (“A”) comprising an antigen-binding region that specifically binds to human V-domain Ig inhibitor of T cell activation (human VISTA); When administered to a subject, VISTA-expressing immune cells, optionally monocytes, myeloid cells, T cells, Tregs, NK cells, neutrophils, dendritic cells, eosinophils, macrophages, NK cells, and endothelium. Uses, agents, compositions according to any of the foregoing, which are preferentially delivered to one or more of the cells, resulting in functional internalization of the anti-inflammatory agent into one or more of said immune cells. It is a specific object of the present invention to provide products or methods.

An anti-human VISTA antibody or antibody fragment that preferentially binds to VISTA-expressing cells at physiological pH (≈7.5), wherein the ADC optionally has a pK of at most 70 hours in human VISTA knock-in rodents. It is a particular object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising:

If the ADC is maintained at physiological pH in cynomolgus monkeys or humans for at most 3.5 ± 0.5 days, more typically at most 48 hours, at most 36 hours, at most 24 hours, at most A specific aspect of the invention is to provide a use, medicament, composition or method according to any of the foregoing, comprising an anti-human VISTA antibody or antibody fragment having a pK of 18 hours, or at most 12 hours. It is a purpose.

an anti-human VISTA, wherein the ADC has a pK of at most 2.8 or 2.3 or 1.5 days, or 1 day, or 12 hours or 8 hours ± 0.5 days in cynomolgus monkeys or humans at physiological pH; It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising an antibody or antibody fragment.

A use, medicament, composition or method according to any of the foregoing, wherein the ADC comprises an anti-human VISTA antibody or antibody fragment having a pK of at most 6-12 hours in human VISTA rodents at physiological pH. It is a specific object of the present invention to provide.

ADCs are VISTA-expressing immune cells, optionally T cells, CD4 or CD8 T cells, Tregs, NK cells, neutrophils, monocytes, myeloid cells, dendritic cells, macrophages, eosinophils, and endothelial cells. an anti-human VISTA antibody or antibody comprising a linker that is cleaved upon internalization of the ADC into one or more of the antibodies, resulting in the release of a therapeutically effective amount of an anti-inflammatory agent (glucocorticoid agonist) within the immune cell. It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising a fragment.

The ADC comprises an anti-human VISTA antibody or antibody fragment, and the anti-VISTA antibody or antigen-binding fragment is present at physiological pH (about pH 7.5) for about 2.3 days in a primate, optionally a cynomolgus monkey. It is a specific object of the invention to provide a use, medicament, composition or method according to any of the foregoing, having an in vivo serum half-life.

The ADC comprises an anti-human VISTA antibody or antibody fragment, and the anti-VISTA antibody or antigen-binding fragment is present at physiological pH (approximately pH 7.5) for up to 70 hours, up to 60 hours, up to 50 hours in human VISTA knock-in rodents. 40 hours or less, 30 hours or less, 24 hours or less, 22 to 24 hours or less, 20 to 22 hours or less, 18 to 20 hours or less, 16 to 18 hours or less, 14 to 16 hours or less, 12 to 14 hours or less, 10 to 12 hours or less, 8 to 10 hours or less, 6 to 8 hours or less, 4 to 6 hours or less, 2 to 4 hours or less, 1 to 2 hours or less, 0.5 to 1.0 hours or less, or 0.1 It is a specific object of the present invention to provide a use, medicament, composition, or method according to any of the foregoing, having an in vivo serum half-life in serum of ˜0.5 hours or less.

When the ADC comprises an anti-human VISTA antibody or antibody fragment, and the PD/PK ratio of the ADC is used in vivo, in a human VISTA knock-in rodent, or in a human or non-human primate, optionally in a cynomolgus monkey. , at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, It is a specific object of the invention to provide a use, medicament, composition or method according to any of the foregoing, wherein the ratio is 14:1 or higher.

the ADC comprises an anti-human VISTA antibody or antibody fragment, and the PD of the ADC is at least 4, 5, in any one of rodents, or human or non-human primates, optionally cynomolgus monkeys; 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, 2 to 4 weeks, 1 month, or longer, use, agent, composition, or method according to any of the foregoing. It is a specific object of the present invention to provide.

the ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell expressed antigen, such as an anti-human VISTA antibody or antibody fragment, the antibody comprising an Fc region with impaired or intact FcR binding; It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing.

An anti-human VISTA antibody or antibody, wherein the ADC is an antibody or antibody fragment that targets a human immune cell-expressed antigen, e.g., an anti-human VISTA antibody or antibody comprising a human IgG1, IgG2, IgG3, or IgG4 Fc region with impaired or intact FcR binding. It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising a fragment.

Any of the foregoing, wherein the ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell expressed antigen, e.g. an anti-human VISTA antibody or antibody fragment comprising a human IgG1 Fc region with impaired FcR binding. It is a specific object of the present invention to provide uses, medicaments, compositions or methods for.

A human or non-human primate in which the ADC is an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, such as a human or non-human primate that has been modified to impair or eliminate binding to at least two native human Fc gamma receptors. It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising an anti-human VISTA antibody or antibody fragment containing a similar constant or Fc region.

The ADC is an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, such as the following FcRs: hFcγRI (CD64), FcyRIIA or hFcyRIIB, (CD32 or CD32A), and FcγRllA (CD16A) or FcγRllB (CD16B). an anti-human comprising a human or non-human primate constant or Fc region that has been modified to impair or eliminate binding to any one, two, three, four, or all five of It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising a VISTA antibody or antibody fragment.

The ADC is an antibody or antibody fragment that specifically binds to a human immune cell expressed antigen, e.g. It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising an anti-human VISTA antibody or antibody fragment comprising an IgG2 kappa backbone.

The ADC is an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, such as an IgG1 having a L234A/L235A silencing mutation in the Fc region, optionally a mutation that impairs complement (C1Q) binding. It is a specific object of the present invention to provide a use, medicament, composition or method according to any of the foregoing, comprising an anti-human VISTA antibody or antibody fragment comprising a /kappa backbone.

The ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell expressed antigen, such as a human IgG1/kappa backbone, optionally having L234A/L235A silencing mutations and E269R and E233A mutations in the Fc region. It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising an anti-human VISTA antibody or antibody fragment comprising:

The ADC includes an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, such as an anti-human VISTA antibody or antibody fragment, and binding of the antibody or antigen-binding fragment to an immune cell is a binding antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen. It is a particular object of the present invention to provide a use, medicament, composition or method according to any of the foregoing that does not directly agonize or antagonize antigen-mediated effects, such as VISTA-mediated effects on immunity. be.

The ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, e.g., an anti-human VISTA antibody or antibody fragment comprising a human IgG1, IgG2, IgG3, or IgG4 Fc region, and endogenous FcR binding is It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, which is undamaged.

Any of the foregoing, wherein the ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell expressed antigen, such as an anti-human VISTA antibody or antibody fragment comprising a native (unmodified) human IgG2 Fc region. It is a specific object of the present invention to provide uses, medicaments, compositions, or methods.

The ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, such as an anti-human VISTA antibody or antibody fragment, and the antibody or antigen-binding fragment is subjected to surface plasmon resonance (SPR) at 24°C or 37°C. The use, medicament, composition according to any of the foregoing, comprising a KD in the range of 0.0001 nM to 10.0 nM, 0.001 to 1.0 nM, or 0.01 to 0.7, as determined by It is a specific object of the present invention to provide a method.

The ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, such as an anti-human VISTA antibody or antibody fragment, and the antibody or antigen-binding fragment is subjected to surface plasmon resonance (SPR) at 24°C or 37°C. It is a specific object of the invention to provide a use, medicament, composition or method according to any of the foregoing, comprising a KD of 0.13 to 0.64 nM as determined by:

The ADC optionally comprises an antibody or antibody fragment that specifically binds to a human immune cell expressed antigen, such as an anti-human VISTA antibody or antibody fragment, and the drug-antibody ratio is in the range of 1:1 to 12:1. It is a particular object of the invention to provide a use, medicament, composition or method according to any of the foregoing, wherein the method is:

The ADC optionally comprises an anti-human VISTA antibody or antibody fragment, and the drug-antibody ratio is in the range of 2-12:1, 2-8:1, 4-8:1, or 6-8:1. It is a specific object of the invention to provide certain uses, medicaments, compositions or methods as described above.

the ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, such as an anti-human VISTA antibody or antibody fragment, and the drug-antibody ratio is about 8:1 (n=8), or It is a specific object of the invention to provide a use, medicament, composition or method according to any of the foregoing, wherein the ratio is about 4:1 (n=4).

The ADC internalizes antibodies or antibody fragments that specifically bind to human immune cell-expressed antigens, e.g., monocytes, myeloid cells, T cells, Tregs, macrophages, and neutrophils. It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising a human VISTA antibody or antibody fragment.

The use, medicament, according to any of the foregoing, wherein the ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, such as an anti-human VISTA antibody or antibody fragment that does not appreciably internalize B cells. It is a specific object of the present invention to provide a composition, composition, or method.

When an ADC is administered to a subject in need of an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, e.g., the same dose of antibody administered in naked (unconjugated) form Uses according to any of the foregoing, including anti-human VISTA antibodies or antibody fragments that promote efficacy and/or reduce adverse side effects, such as toxicity, associated with anti-inflammatory agents as compared to inflammatory agents. It is a specific object of the present invention to provide medicaments, compositions, or methods.

The ADC optionally comprises an antibody or antibody fragment that specifically binds to a human immune cell expressed antigen, such as an anti-human VISTA antibody or antibody fragment, and the glucocorticoid optionally binds via an interchain disulfide. It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, conjugated to an antibody or antigen binding fragment.

The use, medicament, composition according to any of the foregoing, wherein the ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell expressed antigen, e.g. an anti-human VISTA antibody or antibody fragment comprising an esterase-sensitive linker. It is a specific object of the present invention to provide a method.

The ADC comprises an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, such as an anti-human VISTA antibody or antibody fragment, and the cleavable linker is capable of acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage. A specific aspect of the invention is to provide a use, medicament, composition, or method according to any of the foregoing, which is susceptible to one or more of the following: cleavage, esterase-induced cleavage, and disulfide bond cleavage. It is a purpose.

The ADC contains an antibody or antibody fragment that specifically binds to a human immune cell-expressed antigen, such as an anti-human VISTA antibody or antibody fragment, and the anti-VISTA antigen-binding fragment contained in the ADC is Fab, F(ab')2 It is a particular object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising scFv antibody fragments.

The ADC comprises an anti-human VISTA antibody or antibody fragment, wherein the anti-VISTA antibody or antibody fragment contained therein comprises the same CDRs as an antibody having the sequence of FIG. 8, 10, or 12, or optionally ,
(i) comprising the V _H CDRs of SEQ ID NOs: 100, 101, and 102 and the V _L CDRs of SEQ ID NOs: 103, 104, and 105;
(ii) comprising the V _H CDRs of SEQ ID NOs: 110, 111, and 112 and the V _L CDRs of SEQ ID NOs: 113, 114, and 115;
(iii) comprising the V _H CDRs of SEQ ID NOs: 120, 121, and 122 and the V _L CDRs of SEQ ID NOs: 123, 124, and 125;
(iv) comprising the V _H CDRs of SEQ ID NOs: 130, 131, and 132 and the V _L CDRs of SEQ ID NOs: 133, 134, and 135;
(v) comprising the V _H CDRs of SEQ ID NOs: 140, 141, and 142 and the V _L CDRs of SEQ ID NOs: 143, 144, and 145;
(vi) comprising the V _H CDRs of SEQ ID NOs: 150, 151, and 152 and the V _L CDRs of SEQ ID NOs: 153, 154, and 155;
(vii) comprising the V _H CDRs of SEQ ID NOs: 160, 161, and 162 and the V _L CDRs of SEQ ID NOs: 163, 164, and 165;
(viii) comprising the V _H CDRs of SEQ ID NOs: 170, 171, and 172 and the V _L CDRs of SEQ ID NOs: 173, 174, and 175;
(ix) those comprising the V _H CDRs of SEQ ID NOs: 180, 181, and 182 and the V _L CDRs of SEQ ID NOs: 183, 184, and 185;
(x) comprising the V _H CDRs of SEQ ID NOs: 190, 191, and 192 and the V _L CDRs of SEQ ID NOs: 193, 194, and 195;
(xi) comprising the V _H CDRs of SEQ ID NOs: 200, 201, and 202 and the V _L CDRs of SEQ ID NOs: 203, 204, and 205;
(xii) comprising the V _H CDRs of SEQ ID NOs: 210, 211, and 212 and the V _L CDRs of SEQ ID NOs: 213, 214, and 215;
(xiii) those comprising the V _H CDRs of SEQ ID NOs: 220, 221, and 222 and the V _L CDRs of SEQ ID NOs: 223, 224, and 225;
(xiv) those comprising the V _H CDRs of SEQ ID NOs: 230, 231, and 232 and the V _L CDRs of SEQ ID NOs: 233, 234, and 235;
(xv) those comprising the V _H CDRs of SEQ ID NOs: 240, 241, and 242 and the V _L CDRs of SEQ ID NOs: 243, 244, and 245;
(xvi) those comprising the V _H CDRs of SEQ ID NOs: 250, 251, and 252 and the V _L CDRs of SEQ ID NOs: 253, 254, and 255;
(xvii) comprising the VH CDRs of SEQ ID NOs: 260, 261, and 262 and the V _L CDRs of SEQ ID NOs: 263, 264, and 265;
(xviii) those comprising the V _H CDRs of SEQ ID NOs: 270, 271, and 272 and the V _L CDRs of SEQ ID NOs: 273, 274, and 275;
(xix) those comprising the V _H CDRs of SEQ ID NOs: 280, 281, and 282 and the V _L CDRs of SEQ ID NOs: 283, 284, and 285;
(xx) those comprising the V _H CDRs of SEQ ID NOs: 290, 291, and 292 and the V _L CDRs of SEQ ID NOs: 293, 294, and 295;
(xxi) those comprising the V _H CDRs of SEQ ID NOs: 300, 301, and 302 and the V _L CDRs of SEQ ID NOs: 303, 304, and 305;
(xxii) comprising the V _H CDRs of SEQ ID NOs: 310, 311, and 312 and the V _L CDRs of SEQ ID NOs: 313, 314, and 315;
(xxiii) comprising the V _H CDRs of SEQ ID NOs: 320, 321, and 322 and the V _L CDRs of SEQ ID NOs: 323, 324, and 325;
(xxiv) those comprising the V _H CDRs of SEQ ID NOs: 330, 331, and 332 and the V _L CDRs of SEQ ID NOs: 333, 334, and 335;
(xxv) those comprising the V _H CDRs of SEQ ID NOs: 340, 341, and 342 and the V _L CDRs of SEQ ID NOs: 343, 344, and 345;
(xxvi) those comprising the V _H CDRs of SEQ ID NOs: 350, 351, and 352 and the V _L CDRs of SEQ ID NOs: 353, 354, and 355;
(xxvii) those comprising the V _H CDRs of SEQ ID NOs: 360, 361, and 362 and the V _L CDRs of SEQ ID NOs: 363, 364, and 365;
(xxviii) comprising the V _H CDRs of SEQ ID NOs: 370, 371, and 372 and the V _L CDRs of SEQ ID NOs: 373, 374, and 375;
(xxix) comprising the V _H CDRs of SEQ ID NOs: 380, 381, and 382 and the V _L CDRs of SEQ ID NOs: 383, 384, and 385;
(xxx) comprising the V _H CDRs of SEQ ID NOs: 390, 391, and 392 and the V _L CDRs of SEQ ID NOs: 393, 394, and 395;
(xxxi) those comprising the V _H CDRs of SEQ ID NOs: 400, 401, and 402 and the V _L CDRs of SEQ ID NOs: 403, 404, and 405;
(xxxii) comprising the V _H CDRs of SEQ ID NOs: 410, 411, and 412 and the V _L CDRs of SEQ ID NOs: 413, 414, and 415;
(xxxiii) those comprising the V _H CDRs of SEQ ID NOs: 420, 421, and 422 and the V _L CDRs of SEQ ID NOs: 423, 424, and 425;
(xxxiv) those comprising the V _H CDRs of SEQ ID NOs: 430, 431, and 432 and the V _L CDRs of SEQ ID NOs: 433, 434, and 435;
(xxxv) those comprising the V _H CDRs of SEQ ID NOs: 440, 441, and 442 and the V _L CDRs of SEQ ID NOs: 443, 444, and 445;
(xxxvi) those comprising the V _H CDRs of SEQ ID NOs: 450, 451, and 452 and the V _L CDRs of SEQ ID NOs: 453, 454, and 455;
(xxxvii) comprising the V _H CDRs of SEQ ID NOs: 460, 461, and 462 and the V _L CDRs of SEQ ID NOs: 463, 464, and 465;
(xxxviii) comprising the V _H CDRs of SEQ ID NOs: 470, 471, and 472 and the V _L CDRs of SEQ ID NOs: 473, 474, and 475;
(xxxix) comprising the V _H CDRs of SEQ ID NOs: 480, 481, and 482 and the V _L CDRs of SEQ ID NOs: 483, 484, and 485;
(xl) those comprising the V _H CDR polypeptides of SEQ ID NOs: 490, 491, and 492 and the VL CDR polypeptides of SEQ ID NOs: 493, 494, and 495;
(xli) those comprising the V _H CDR polypeptides of SEQ ID NOs: 500, 501, and 502 and the VL CDR polypeptides of SEQ ID NOs: 503, 504, and 505;
(xlii) comprising the V _H CDR polypeptides of SEQ ID NOs: 510, 511, and 512 and the VL CDR polypeptides of SEQ ID NOs: 513, 514, and 515;
(xliii) comprising the V _H CDR polypeptides of SEQ ID NOs: 520, 521, and 522 and the VL CDR polypeptides of SEQ ID NOs: 523, 524, and 525;
(xliv) comprising the V _H CDR polypeptides of SEQ ID NOs: 530, 531, and 532, and the VL CDR polypeptides of SEQ ID NOs: 533, 534, and 535;
(xlv) comprising the V _H CDR polypeptides of SEQ ID NOs: 540, 541, and 542, and the VL CDR polypeptides of SEQ ID NOs: 543, 544, and 545;
(xlvi) comprising the V _H CDR polypeptides of SEQ ID NOs: 550, 551, and 552, and the VL CDR polypeptides of SEQ ID NOs: 553, 554, and 555;
(xlvii) those comprising the V _H CDRs of SEQ ID NOs: 560, 561, and 562 and the V _L CDRs of SEQ ID NOs: 563, 564, and 565;
(xlviii) those comprising the V _H CDRs of SEQ ID NOs: 570, 571, and 572 and the V _L CDRs of SEQ ID NOs: 573, 574, and 575;
(xlix) comprising the V _H CDRs of SEQ ID NOs: 580, 581, and 582 and the V _L CDRs of SEQ ID NOs: 583, 584, and 585;
(l) comprising the V _H CDRs of SEQ ID NOs: 590, 591, and 592 and the V _L CDRs of SEQ ID NOs: 593, 594, and 595;
(li) comprising the V _H CDRs of SEQ ID NOs: 600, 601, and 602 and the V _L CDRs of SEQ ID NOs: 603, 604, and 605;
(lii) comprising the V _H CDRs of SEQ ID NOs: 610, 611, and 612 and the V _L CDRs of SEQ ID NOs: 613, 614, and 615;
(liii) those comprising the V _H CDRs of SEQ ID NOs: 620, 621, and 622 and the V _L CDRs of SEQ ID NOs: 623, 624, and 625;
(liv) those comprising the V _H CDRs of SEQ ID NOs: 630, 631, and 632 and the V _L CDRs of SEQ ID NOs: 633, 634, and 635;
(lv) those comprising the V _H CDRs of SEQ ID NOs: 640, 641, and 642 and the V _L CDRs of SEQ ID NOs: 643, 644, and 645;
(lvi) those comprising the V _H CDRs of SEQ ID NOs: 650, 651, and 652 and the V _L CDRs of SEQ ID NOs: 653, 654, and 655;
(lvii) comprising the V _H CDRs of SEQ ID NOs: 660, 661, and 662 and the V _L CDRs of SEQ ID NOs: 663, 664, and 665;
(lviii) those comprising the V _H CDRs of SEQ ID NOs: 670, 671, and 672 and the V _L CDRs of SEQ ID NOs: 673, 674, and 675;
(lix) those comprising the V _H CDRs of SEQ ID NOs: 680, 681, and 682 and the V _L CDRs of SEQ ID NOs: 683, 684, and 685;
(lx) comprising the V _H CDRs of SEQ ID NOs: 690, 691, and 692 and the V _L CDRs of SEQ ID NOs: 693, 694, and 695;
(lxi) those comprising the V _H CDRs of SEQ ID NOs: 700, 701, and 702 and the V _L CDRs of SEQ ID NOs: 703, 704, and 705;
(lxii) comprising the V _H CDRs of SEQ ID NOs: 710, 711, and 712 and the V _L CDRs of SEQ ID NOs: 713, 714, and 715;
(lxiii) those comprising the V _H CDRs of SEQ ID NOs: 720, 721, and 722 and the V _L CDRs of SEQ ID NOs: 723, 724, and 725;
(lxiv) those comprising the V _H CDRs of SEQ ID NOs: 730, 731, and 732 and the V _L CDRs of SEQ ID NOs: 733, 734, and 735;
(lxv) comprising the V _H CDRs of SEQ ID NOs: 740, 741, and 742 and the V _L CDRs of SEQ ID NOs: 743, 744, and 745;
(lxvi) comprising the V _H CDRs of SEQ ID NOs: 750, 751, and 752 and the V _L CDRs of SEQ ID NOs: 753, 754, and 755;
(lxvii) comprising the V _H CDRs of SEQ ID NOs: 760, 761, and 762 and the V _L CDRs of SEQ ID NOs: 763, 764, and 765;
(lxviii) those comprising the V _H CDRs of SEQ ID NOs: 770, 771, and 772 and the V _L CDRs of SEQ ID NOs: 773, 774, and 775;
(lxix) comprising the V _H CDRs of SEQ ID NOs: 780, 781, and 782 and the V _L CDRs of SEQ ID NOs: 783, 784, and 785;
(lxx) comprising the V _H CDRs of SEQ ID NOs: 790, 791, and 792 and the V _L CDRs of SEQ ID NOs: 793, 794, and 795;
(lxxi) those comprising the V _H CDRs of SEQ ID NOs: 800, 801, and 802 and the V _L CDRs of SEQ ID NOs: 803, 804, and 805;
(lxxii) the use according to any of the foregoing, which is selected from the V _H CDRs of SEQ ID NOs: 810, 811, and 812, and the V _L CDRs of SEQ ID NOs: 813, 814, and 815; It is a specific object of the present invention to provide a medicament, composition, or method.

Provided is an antibody drug conjugate (ADC) according to any of the foregoing, wherein the ADC comprises an anti-VISTA antibody or antibody fragment comprising the same CDR as any one of VSTB92, VSTB56, VSTB95, VSTB103, and VSTB66. It is a specific objective of the present invention to do so.

An antibody drug conjugate (ADC) according to any of the foregoing, wherein the ADC comprises an anti-VISTA antibody or antibody fragment comprising a VH polypeptide and a VL polypeptide, each of the following VH and VL polypeptides: An antibody comprising
(i) comprising a V _H polypeptide of SEQ ID NO: 106 identity and a V _L polypeptide of SEQ ID NO: 108;
(ii) comprising a V _H polypeptide of SEQ ID NO: 116 and a V _L polypeptide of SEQ ID NO: 118;
(iii) comprising a V _H polypeptide of SEQ ID NO: 126 and a V _L polypeptide of SEQ ID NO: 128;
(iv) comprising a V _H polypeptide of SEQ ID NO: 136 and a V _L polypeptide of SEQ ID NO: 138;
(v) comprising a V _H polypeptide of SEQ ID NO: 146 and a V _L polypeptide of SEQ ID NO: 148;
(vi) comprising a V _H polypeptide of SEQ ID NO: 156 and a V _L polypeptide of SEQ ID NO: 158;
(vii) comprising a V _H polypeptide of SEQ ID NO: 166 and a V _L polypeptide of SEQ ID NO: 168;
(viii) comprising a V _H polypeptide of SEQ ID NO: 176 and a V _L polypeptide of SEQ ID NO: 178;
(ix) comprising the V _H polypeptide of SEQ ID NO: 186 and the V _L polypeptide of SEQ ID NO: 188;
(x) one comprising the V _H polypeptide of SEQ ID NO: 196 and the V _L polypeptide of SEQ ID NO: 198;
(xi) comprising the V _H polypeptide of SEQ ID NO: 206 and the V _L polypeptide of SEQ ID NO: 208;
(xii) comprising the V _H polypeptide of SEQ ID NO: 216 and the V _L polypeptide of SEQ ID NO: 218;
(xiii) comprising a V _H polypeptide of SEQ ID NO: 226 and a V _L polypeptide of SEQ ID NO: 228;
(xiv) comprising a V _H polypeptide of SEQ ID NO: 236 and a V _L polypeptide of SEQ ID NO: 238;
(xv) comprising a V _H polypeptide of SEQ ID NO: 246 and a V _L polypeptide of SEQ ID NO: 248;
(xvi) comprising a V _H polypeptide of SEQ ID NO: 256 and a V _L polypeptide of SEQ ID NO: 258;
(xvii) comprising a V _H polypeptide of SEQ ID NO: 266 and a V _L polypeptide of SEQ ID NO: 268;
(xviii) comprising a V _H polypeptide of SEQ ID NO: 276 and a VL polypeptide of SEQ ID NO: 278;
(xix) comprising a V _H polypeptide of SEQ ID NO: 286 and a V _L polypeptide of SEQ ID NO: 288;
(xx) comprising a V _H polypeptide of SEQ ID NO: 296 and a V _L polypeptide of SEQ ID NO: 298;
(xxi) one comprising a V _H polypeptide of SEQ ID NO: 306 and a V _L polypeptide of SEQ ID NO: 308;
(xxii) comprising a V _H polypeptide of SEQ ID NO: 316 and a V _L polypeptide of SEQ ID NO: 318;
(xxiii) comprising a V _H polypeptide of SEQ ID NO: 326 and a V _L polypeptide of SEQ ID NO: 328;
(xxiv) comprising a V _H polypeptide of SEQ ID NO: 336 and a V _L polypeptide of SEQ ID NO: 338;
(xxv) comprising a V _H polypeptide of SEQ ID NO: 346 and a V _L polypeptide of SEQ ID NO: 348;
(xxvi) comprising a V _H polypeptide of SEQ ID NO: 356 and a V _L polypeptide of SEQ ID NO: 358;
(xxvii) comprising a V _H polypeptide of SEQ ID NO: 366 and a V _L polypeptide of SEQ ID NO: 368;
(xxviii) comprising a V _H polypeptide of SEQ ID NO: 376 and a V _L polypeptide of SEQ ID NO: 378;
(xxix) comprising a V _H polypeptide of SEQ ID NO: 386 and a V _L polypeptide of SEQ ID NO: 388;
(xxx) comprising a V _H polypeptide of SEQ ID NO: 396 and a V _L polypeptide of SEQ ID NO: 398;
(xxxi) comprising a V _H polypeptide of SEQ ID NO: 406 and a V _L polypeptide of SEQ ID NO: 408;
(xxxii) comprising a V _H polypeptide of SEQ ID NO: 416 and a V _L polypeptide of SEQ ID NO: 418;
(xxxiii) comprising a V _H polypeptide of SEQ ID NO: 426 and a V _L polypeptide of SEQ ID NO: 428;
(xxxiv) comprising a V _H polypeptide of SEQ ID NO: 436 and a V _L polypeptide of SEQ ID NO: 438;
(xxxv) comprising a V _H polypeptide of SEQ ID NO: 446 and a V _L polypeptide of SEQ ID NO: 448;
(xxxvi) comprising a V _H polypeptide of SEQ ID NO: 456 and a V _L polypeptide of SEQ ID NO: 458;
(xxxvii) comprising a V _H polypeptide of SEQ ID NO: 466 and a V _L polypeptide of SEQ ID NO: 468;
(xxxviii) comprising a V _H polypeptide of SEQ ID NO: 476 and a V _L polypeptide of SEQ ID NO: 478;
(xxxix) comprising a V _H polypeptide of SEQ ID NO: 486 and a V _L polypeptide of SEQ ID NO: 488;
(xl) comprising a V _H polypeptide of SEQ ID NO: 496 and a V _L polypeptide of SEQ ID NO: 498;
(xli) one comprising a V _H polypeptide of SEQ ID NO: 506 and a V _L polypeptide of SEQ ID NO: 508;
(xlii) comprising a V _H polypeptide of SEQ ID NO: 516 and a V _L polypeptide of SEQ ID NO: 518;
(xliii) comprising a V _H polypeptide of SEQ ID NO: 526 and a V _L polypeptide of SEQ ID NO: 528;
(xliv) comprising a V _H polypeptide of SEQ ID NO: 536 and a V _L polypeptide of SEQ ID NO: 533, 534, and 535;
(xlv) comprising a V _H polypeptide of SEQ ID NO: 546 and a V _L polypeptide of SEQ ID NO: 548;
(xlvi) comprising a V _H polypeptide of SEQ ID NO: 556 and a V _L polypeptide of SEQ ID NO: 558;
(xlvii) comprising a V _H polypeptide of SEQ ID NO: 566 and a V _L polypeptide of SEQ ID NO: 568;
(xlviii) comprising a V _H polypeptide of SEQ ID NO: 576 and a V _L polypeptide of SEQ ID NO: 578;
(xlix) comprising a V _H polypeptide of SEQ ID NO: 586 and a V _L polypeptide of SEQ ID NO: 588;
(l) comprising a V _H polypeptide of SEQ ID NO: 596 and a V _L polypeptide of SEQ ID NO: 598;
(li) comprising a V _H polypeptide of SEQ ID NO: 606 and a V _L polypeptide of SEQ ID NO: 608;
(lii) comprising a V _H polypeptide of SEQ ID NO: 616 and a V _L polypeptide of SEQ ID NO: 618;
(liii) comprising a V _H polypeptide of SEQ ID NO: 626 and a V _L polypeptide of SEQ ID NO: 628;
(liv) comprising the V _H polypeptide of SEQ ID NO: 636 and the V _L polypeptide of SEQ ID NO: 638;
(lv) comprising a V _H polypeptide of SEQ ID NO: 646 and a V _L polypeptide of SEQ ID NO: 648;
(lvi) comprising a V _H polypeptide of SEQ ID NO: 656 and a V _L polypeptide of SEQ ID NO: 658;
(lvii) comprising a V _H polypeptide of SEQ ID NO: 666 and a V _L polypeptide of SEQ ID NO: 668;
(lviii) comprising a V _H polypeptide of SEQ ID NO: 676 and a V _L polypeptide of SEQ ID NO: 678;
(lix) comprising a V _H polypeptide of SEQ ID NO: 686 and a V _L polypeptide of SEQ ID NO: 688;
(lx) comprising a V _H polypeptide of SEQ ID NO: 696 and a V _L polypeptide of SEQ ID NO: 698;
(lxi) one comprising a V _H polypeptide of SEQ ID NO: 706 and a V _L polypeptide of SEQ ID NO: 708;
(lxii) comprising a V _H polypeptide of SEQ ID NO: 716 and a V _L polypeptide of SEQ ID NO: 718;
(lxiii) comprising a V _H polypeptide of SEQ ID NO: 726 and a V _L polypeptide of SEQ ID NO: 728;
(lxiv) comprising a V _H polypeptide of SEQ ID NO: 736 and a V _L polypeptide of SEQ ID NO: 738;
(lxv) comprising a V _H polypeptide of SEQ ID NO: 746 and a V _L polypeptide of SEQ ID NO: 748;
(lxvi) comprising a V _H polypeptide of SEQ ID NO: 756 and a V _L polypeptide of SEQ ID NO: 758;
(lxvii) comprising a V _H polypeptide of SEQ ID NO: 766 and a V _L polypeptide of SEQ ID NO: 768;
(lxviii) comprising a V _H polypeptide of SEQ ID NO: 776 and a V _L polypeptide of SEQ ID NO: 778;
(lxix) comprising a V _H polypeptide of SEQ ID NO: 786 and a V _L polypeptide of SEQ ID NO: 788;
(lxx) comprising a V _H polypeptide of SEQ ID NO: 796 and a V _L polypeptide of SEQ ID NO: 798;
(lxxxi) those comprising the V _H polypeptide of SEQ ID NO: 806 and the V _L polypeptide of SEQ ID NO: 808; and (lxxii) those comprising the V _H polypeptide of SEQ ID NO: 816 and the V _L polypeptide of SEQ ID NO: 818. It is a specific object of the invention to provide an ADC that possesses at least 90%, 95%, or 100% sequence identity with an antibody containing an antibody, and in which the CDRs are unmodified.

The use according to any of the foregoing, wherein the ADC comprises an anti-human VISTA antibody or antibody fragment, and the anti-VISTA antibody or antibody fragment comprises the same variable region as one of VSTB92, VSTB56, VSTB95, VSTB103, and VSTB66. It is a specific object of the present invention to provide medicaments, compositions, or methods.

The ADC optionally comprises an anti-human VISTA antibody or antibody fragment having the CDRs or variable sequences of the antibody of FIG. A specific aspect of the invention is to provide a use, medicament, composition or method according to any of the foregoing, comprising a human IgG2 kappa backbone with /P238S/H268A/V309L/A330S/P331S silencing mutations. It is a purpose.

The ADC optionally comprises an anti-human VISTA antibody or antibody fragment having the CDRs or variable sequences of the antibody of FIG. It is a specific object of the invention to provide a use, medicament, composition or method as described in any of the foregoing, comprising a human IgG1/kappa scaffold with a silencing mutation.

The glucocorticosteroid agonist or linker conjugate is conjugated via its interchain disulfide to an antibody or antibody fragment, such as an anti-human VISTA antibody or antibody fragment, that specifically binds to a human immune cell expressed antigen. It is a specific object of the present invention to provide an ADC according to any of the foregoing.

There is provided a pharmaceutical composition comprising a therapeutically effective amount of at least one antibody drug conjugate (ADC) or steroid agonist or steroid linker as described above, and a pharmaceutically acceptable carrier. , is a specific object of the present invention.

It is a particular object of the present invention to provide a composition according to any of the foregoing, which is administrable via injection route, optionally intravenous, intramuscular, intrathecal or subcutaneous administration. It is.

It is a specific object of the invention to provide a composition according to any of the foregoing, which is administrable subcutaneously.

A glucocorticosteroid agonist, linker, according to any of the foregoing, providing subcutaneous administration selected from the group consisting of a syringe, an injection device, an infusion pump, an injector pen, a needleless device, an autoinjector, and a subcutaneous patch delivery system. It is a specific object of the present invention to provide devices containing conjugates, ADCs, compositions, or agents.

A device that delivers a fixed dose of a glucocorticoid receptor agonist or functional derivative thereof to a patient and optionally further comprises instructions informing the patient of the method and dosing regimen for administering the ADC composition contained therein. It is a specific object of the present invention to provide.

administering to a patient in need of treatment and/or prophylaxis at least one antibody drug conjugate (ADC) or steroid or composition according to any of the foregoing, wherein said composition It is a particular object of the invention to provide a method of treatment and/or prophylaxis, which may be present in a device as described in .

In human subjects, for the treatment of allergy, autoimmunity, transplantation, gene therapy, inflammation, GVHD, or sepsis, or to treat or treat inflammatory, autoimmune, or allergic side effects associated with any of the foregoing conditions. It is a specific object of the present invention to provide a method of treatment and/or prophylaxis according to any of the above, for use in preventing.

Patients with rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, sweat gland abscess, uveitis, Behcet's disease, spinal joint It is a particular object of the invention to provide a method of treatment and/or prophylaxis according to any of the foregoing, including a condition selected from psoriasis or psoriasis.

The patient has one or more of the following:
(i) a chronic, acute, episodic allergy, inflammation, or inflammatory condition, e.g., chronic, acute, episodic, remitting/relapsing;
(ii) a condition that can be effectively treated only temporarily with high doses of steroids, optionally for which the patient is being treated or is being treated with high doses of steroids; polymyalgia and/or giant cell arteritis,
(iii) Conditions with comorbidities that limit steroid use, optionally diabetes, nonalcoholic steatohepatitis (NASH), morbid obesity, avascular necrosis/osteonecrosis (AVN), glaucoma, steroid-induced high blood pressure, severe skin fragility, and/or osteoarthritis;
(iv) conditions for which safe long-term therapeutic agents are available, but where induction for several months with high doses of steroids is desired; optionally, induction for several months with high doses of steroids is therapeutic; AAV, polymyositis, dermatomyositis, lupus, inflammatory lung disease, autoimmune hepatitis, inflammatory bowel disease, immune thrombocytopenia, autoimmune hemolytic anemia, gout patients,
(v) skin conditions requiring short-term/long-term treatment, optionally conditions of uncertain treatment or duration, and/or conditions for which there are no effective alternatives to steroid administration, optionally Stevens Johnson, other severe drug eruption conditions, conditions with extensive contact dermatitis, other severe immune-related skin conditions such as PG, LCV, erythroderma, and the like;
(vi) conditions treated with high-dose corticosteroids for flare-ups/relapses, optionally COPD, asthma, lupus, gout, pseudogout;
(vii) immune-related neurological diseases such as small fiber neuropathy, MS (subset), chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, and the like;
(viii) optionally hematology/oncology indications for which high doses of steroids are therapeutically warranted or beneficial;
(ix) ophthalmological conditions, optionally uveitis, iritis, scleritis, and the like;
(x) conditions associated with persistent or very long-term adrenal insufficiency or secondary adrenal insufficiency, optionally an iatrogenic Addison's disease crisis;
(xi) conditions often treated with long-term low-dose steroids, optionally lupus, RA, psA, vasculitis, and the like;
(xii) any of the foregoing, including pregnant/lactating women, pediatric patients, optionally pediatric patients with growth defects or cataracts, or special classes of patients such as any combination of the foregoing; It is a specific object of the invention to provide the described methods of treatment and/or prevention.

It is a specific object of the invention to provide a method, medicament or use as described in any of the foregoing, wherein the patient is further treated with another active agent.

The patient has received an immunoinhibitory antibody or fusion protein that targets one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA, and and/or further treated with an immunomodulatory antibody or fusion protein selected from agonistic antibodies or fusion proteins targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28, or ICOS. It is a specific object of the present invention to provide any method of treatment and/or prophylaxis as described.

Immune cells from a patient or donor are contacted with an ADC or steroid as described in any of the foregoing and then a patient in need thereof, e.g. a patient with one or more of the previously identified conditions. It is a specific object of the present invention to provide an ex vivo use of an ADC or steroid as described above, injected into a patient.

A specific aspect of the invention is to provide an ADC according to any of the foregoing, wherein the linker is a positively, negatively or neutrally charged cleavable peptide, optionally esterase cleavable. It is a purpose.

It is a specific object of the present invention to provide an ADC according to any of the foregoing, wherein the drug-antibody ratio is in the range of 1:1 to 12:1 or 1:1 to 10:1.

It is a specific object of the present invention to provide an ADC according to any of the foregoing, wherein the drug-antibody ratio is in the range of 2-8:1, 4-8:1, or 6-8:1. be.

A specific aspect of the invention is to provide an ADC according to any of the foregoing, wherein the drug-antibody ratio drug-antibody ratio is about 8:1 (n=8) or about 4:1 (n=4). It is a purpose.

Among several immune cell types, activated and non-activated monocytes, myeloid cells, B cells, NK cells, T cells, CD4 T cells, CD8 T cells, Tregs, mast cells, eosinophils, dendritic cells. It is a specific object of the present invention to provide an ADC according to any of the foregoing, which internalizes one or more of the following: , mast cells, macrophages, and neutrophils.

It is a specific object of the invention to provide an ADC according to any of the foregoing that does not appreciably internalize activated or non-activated B cells.

glucocorticoid receptor agonist and/or glucocorticoid receptor agonist when administered to a subject in need of treatment compared to the same dose of anti-inflammatory agent administered in naked (unconjugated) form It is a specific object of the present invention to provide an ADC according to any of the foregoing, which reduces the harmful side effects associated with.

It is a specific object of the present invention to provide an ADC according to any of the foregoing, wherein a glucocorticoid receptor agonist is conjugated to the antibody or antigen binding fragment via an interchain disulfide.

It is a specific object of the invention to provide an ADC according to any of the above, comprising an esterase sensitive linker.

An ADC according to any of the foregoing, comprising a cleavable linker susceptible to one or more of acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. It is a specific object of the present invention to provide.

Any of the foregoing, comprising a non-cleavable linker that is substantially resistant to one or more of acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. It is a specific object of the present invention to provide an ADC as described in .

It is a specific object of the present invention to provide an ADC according to any of the foregoing, wherein the anti-VISTA antigen-binding fragment contained in the ADC comprises a Fab, F(ab')2, or scFv antibody fragment. .

administering to a patient in need of treatment and/or prophylaxis at least one antibody drug conjugate (ADC) or composition, which composition may be present in a device as described above. It is a specific object of the present invention to provide a method of treatment and/or prevention.

administering at least one antibody drug conjugate (ADC) or composition to a patient in need of treatment and/or prophylaxis, the composition comprising: administering to a patient in need of treatment and/or prophylaxis, the composition comprising: , inflammation, GVHD, or sepsis, or to treat or prevent inflammatory, autoimmune, or allergic side effects associated with any of the aforementioned conditions. It is a specific object of the present invention to provide therapeutic and/or prophylactic methods that can be present within a device.

administering to a patient in need of treatment and/or prophylaxis at least one antibody drug conjugate (ADC) or composition, wherein the composition is associated with rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, For the treatment of patients with a condition selected from ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, sweat gland abscess, uveitis, Behcet's disease, spondyloarthropathy, or psoriasis It is a particular object of the invention to provide a method of treatment and/or prophylaxis, which may be present in a device as described above.

administering to a patient in need of treatment and/or prophylaxis at least one antibody drug conjugate (ADC) or composition, the composition comprising one or more of the following:
(i) a condition that can be effectively treated only primarily with high doses of steroids, optionally where the patient is being treated with or is being treated with high doses of steroids; polymyalgia and/or giant cell arteritis,
(ii) Conditions with comorbidities that limit steroid use, optionally diabetes, nonalcoholic steatohepatitis (NASH), morbid obesity, avascular necrosis/osteonecrosis (AVN), glaucoma, steroid-induced high blood pressure, severe skin fragility, and/or osteoarthritis;
(iii) conditions for which safe long-term therapeutic agents are available, but where induction for several months with high doses of steroids is desired; optionally, induction for several months with high doses of steroids is therapeutic; AAV, polymyositis, dermatomyositis, lupus, inflammatory lung disease, autoimmune hepatitis, inflammatory bowel disease, immune thrombocytopenia, autoimmune hemolytic anemia, gout patients,
(iv) skin conditions requiring short-term/long-term treatment, optionally conditions of uncertain treatment or duration, and/or conditions for which there are no effective alternatives to steroid administration, optionally Stevens; Johnson, other severe drug eruption conditions, conditions with extensive contact dermatitis, other severe immune-related skin conditions such as PG, LCV, erythroderma, and the like;
(v) conditions treated with high-dose corticosteroids for flare-ups/relapses, optionally COPD, asthma, lupus, gout, pseudogout;
(vi) immune-related neurological diseases such as small fiber neuropathy, MS (subset), chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, and the like;
(vii) optionally, hematology/oncology indications for which high doses of steroids are therapeutically warranted or beneficial;
(viii) ophthalmological conditions, optionally uveitis, iritis, scleritis, and the like;
(ix) conditions associated with persistent or very long-term adrenal insufficiency or secondary adrenal insufficiency, optionally an iatrogenic Addison's disease crisis;
(x) Conditions often treated with long-term, low-dose steroids, optionally lupus, RA, psA, vasculitis, and the like; and (xi) Pregnant/lactating women, pediatric patients, optionally Optionally, therapeutic and/or prophylactic methods may be present in any of the aforementioned devices for the treatment of patients, including special classes of patients, such as pediatric patients with growth disorders or cataracts. It is a specific object of the present invention to provide.

Treatment of a patient in need of treatment and/or prophylaxis comprising administering at least one antibody drug conjugate (ADC) or composition to a patient in need of such treatment, said composition being further treated with another active agent. It is a particular object of the invention to provide a method of treatment and/or prophylaxis, which may be present in any of the above-mentioned devices for.

administering to a patient in need of treatment and/or prophylaxis at least one antibody-drug conjugate (ADC) or composition, wherein the composition comprises CTLA4, PD-1, PDL-1, LAG-3 , TIM-3, BTLA, B7-H4, B7-H3, VISTA, and/or CD40, CD137, OX40, GITR, CD27, CD28, or ICOS. in a device according to any of the foregoing for the treatment of a patient further being treated with an immunomodulatory antibody or fusion protein optionally selected from agonist antibodies or fusion proteins targeting one or more of It is a specific object of the present invention to provide a method of treatment and/or prophylaxis, which may exist in the human body.

administering to a patient in need of treatment and/or prophylaxis at least one antibody drug conjugate (ADC) or composition, wherein the composition The conditions may be present in the device as described in any of the foregoing for the treatment or prevention of indications, optionally severe asthma, giant cell arteritis, ANKA vasculitis, and IBD (colitis and It is a specific object of the present invention to provide a method of treatment and/or prevention, including Crohn's disease.

administering to a patient in need of treatment and/or prophylaxis at least one antibody drug conjugate (ADC) or composition, wherein the composition is associated with rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, For the treatment and prevention of conditions selected from ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, sweat gland abscess, uveitis, Behçet's disease, spondyloarthropathy, or psoriasis. It is a particular object of the invention to provide a method of treatment and/or prophylaxis, which may be present in a device as described above.

Among several immune cell types, myeloid cells, T cells, CD4 T cells, CD8 T cells, Tregs, NK cells, neutrophils, monocytes, B cells, NK cells, myeloid cells, dendritic cells, eosinophils A method for effecting internalization of steroids into one or more of spheres, mast cells, and macrophages, the method comprising administering to a subject an ADC according to any of the foregoing, or injecting said cells ex vivo with said cells. It is a specific object of the present invention to provide a method comprising contacting.

Among myeloid cells, NK cells, B cells, T cells, CD4 T cells, CD8 T cells, Tregs, NK cells, neutrophils, monocytes, myeloid cells, dendritic cells, eosinophils, mast cells, and macrophages A method for effecting internalization of a steroid into one or more of the following, which is accomplished ex vivo, by administering to a subject an ADC according to any of the foregoing, or contacting said cells ex vivo therewith. cells, CD4 T cells, CD8 T cells, Tregs, NK cells, neutrophils, monocytes, myeloid cells, mast cells, dendritic cells, among other immune cell types. A purified or enriched composition comprising one or more specific types of immune cells selected from cells, eosinophils, and macrophages is contacted ex vivo with an ADC as described in any of the foregoing, and then It is a specific object of the present invention to provide a method that can be introduced into a patient in need thereof.

Bone marrow cells, NK cells, B cells, T cells, CD4 T cells, CD8 T cells, Tregs, NK cells, neutrophils, monocytes, mast cells, bone marrow cells, dendritic cells, eosinophils, mast cells, and A method for effecting internalization of a steroid into one or more of macrophages, the method comprising administering to a subject in need of treatment an ADC according to any of the foregoing; Inflammation involving any one or more of NK cells, B cells, T cells, Tregs, NK cells, neutrophils, monocytes, myeloid cells, dendritic cells, mast cells, eosinophils, and macrophages or in accordance with any of the foregoing methods for treating an autoimmune or allergic condition, the present invention provides a method comprising administering an ADC to a subject, or contacting the cell with the cell ex vivo, according to any of the aforementioned methods for treating an autoimmune or allergic condition. It is a specific object of the invention.

Peptide mapping of control VISTA antibody 767-IgG1.3 by trypsin digestion is shown. Determined sequence of 767-IgG1.3 with identified tryptic peptides (A) light chain (85.6% coverage) (B) heavy chain (76.1% coverage) underlined. . The determined sequence of 767-IgG1.3 using Lys-C digestion is shown. In the figure, Lys-C peptides are underlined (A) light chain (63.3% coverage) and (B) heavy chain (76.3% coverage). Includes the results of binding experiments confirming that synthetic control antibodies 767-IgG1.3 and INX 200 exhibit opposite pH-dependent binding properties. Contains the results of binding studies demonstrating that DAR 8 conjugation with linker A does not affect VISTA binding to INX200. Contains the results of binding studies demonstrating that DAR 8 conjugation with linker A does not affect VISTA binding to INX201. Contains the results of binding studies demonstrating that DAR 8 conjugation with linker A does not affect VISTA binding to 767-IgG1.3. Contains the results of a ConA experiment in which different naked and Dex-conjugated anti-VISTA antibodies were administered to female hVISTA knock-in animals, in which G-CSF changes were detected 6 hours after ConA in peripheral blood. Plasma concentrations (SEM; n = 5/group) measured using mouse 7plex (administration: Dex-0.2 = 0.2 mg/Kg, Dex-2 = 2 mg/Kg, INX210 at 10 mg/Kg and INX210A, [INX210A provided a dex payload of 0.2 mg/kg]). Contains results from a ConA study in which male hVISTA knock-in animals were administered different naked and Dex-conjugated anti-VISTA antibodies. In the experiment shown, cytokines change in peripheral blood 6 hours after ConA. Plasma concentrations measured using mouse 7plex (SEM; n=10/group, normal one-way ANOVA compared to ConA only group) (Dosing: Dex at 0.2 or 5 mg/Kg, 10 mg/Kg INX210 and INX210A). Includes results from ConA experiments in which animals were administered different naked and Dex-conjugated anti-VISTA antibodies and cytokine changes were detected in peripheral blood 6 hours after ConA. Plasma concentrations were measured using an ELISA assay (SD; n=6/group; one-way ANOVA compared to ConA only group) (Dose: Dex at 0.02, 0.2, or 2 mg/Kg; INX200A at 10, 5, and 1 mg/Kg). Includes variable heavy and light and constant region sequences and sequence legends for INX200, INX201, and INX210. Exemplary budenoside derivatives are shown. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. CDRs, variable heavy and light sequences, framework sequences, and Contains a sequence listing, including constant domain and epitope information. Includes exemplary steroid structures from those disclosed in Example 3. Includes exemplary steroid structures from those disclosed in Example 3. Includes exemplary steroid structures from those disclosed in Example 3. Includes sequences of exemplary anti-VISTA antibodies and control antibodies disclosed in the Examples. Includes sequences of exemplary anti-VISTA antibodies and control antibodies disclosed in the Examples. Includes sequences of exemplary anti-VISTA antibodies and control antibodies disclosed in the Examples. INX200, and binding studies of 767-IgG1.3 versus human IgG1si. Median fluorescence intensity measured for monocytes incubated with serial dilutions (0-333 nM) of tested antibodies; black dashed line corresponds to autofluorescence of unstained cells; n=1. The percentage of anti-VISTA antibody (INX200) internalized by immune cells is shown. The intracellular pool of cell-bound antibody was plotted over a 60 minute time course. For each data point, fluorescence was normalized to INX200 fluorescence at time 0 minutes. Mean±SD n=2 donors. Contains the results of experiments evaluating the internalization rate of INX200 antibodies. Internalization rates of INX200 antibodies were assessed in monocytes over a 60 minute time course. Anti-CD45 antibodies were not internalized at any time point. Shown as mean±SD, n=2 donors. Includes PK studies of INX200, INX200A versus human IgG1. Plasma concentrations of antibodies (SD; n=5/group) at annotated time points in hVISTA KI mice. Includes PK studies of 767-IgG1.3, 767-IgG1.3A versus human IgG1. Plasma concentrations of antibodies (SD; n=5/group) at annotated time points in hVISTA KI mice. Contains the results of experiments evaluating the efficacy of ADC conjugates according to the invention. Experiments evaluated FKBP5 transcriptional activation after Dex (left) and ADC INX201J (right) treatment in peritoneal resident macrophages and splenic monocytes. Dex (left) effects were evaluated at 4 and 24 hours after a single intraperitoneal (i.p.) injection at 2 mg/Kg. ADC (right) effects were analyzed at 24, 48, 72 and 96 hours after a single intraperitoneal injection at 10 mg/Kg delivering a GC payload of 0.2 mg/Kg. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus PBS control group. Four mice per group were pooled together to generate sufficient material for RNA preparation. Contains the results of in vivo experiments showing that Dex treatment prevents ex vivo induction of inflammatory cytokines in PRM. Dex effects were evaluated 2 hours after a single intraperitoneal injection at 2 mg/Kg. IL-6 and TNFα were assessed in cell supernatants (collected at 1 hour) using mouse 32-plexes (n=4 mice/group; unpaired T-test). Includes results of experiments evaluating the in vivo effects of INX201J or Dex treatment on TNFα in PRM. Results show that INX201J or Dex treatment prevents ex vivo induction of TNFα in PRM. In these experiments, Dex effects were evaluated 2 hours after a single intraperitoneal injection at 2 and 0.2 mg/Kg. INX201J efficacy was evaluated on day 1 (d-1), day 2 (d-2) and day 4 (d-4) after injection at 10 mg/Kg (equivalent to a payload of 0.2 mg/Kg). Cell supernatants were collected at 2 hours. TNFα was measured using ELISA (n=4 mice/group; conventional one-way ANOVA compared to PBS only group). Contains the results of experiments evaluating the long-term effects of exemplary ADCs according to the invention. The results show that all tested ADCs induced long-term effects on ex vivo induction of TNFα and IL-6 during PRM. Dex effects were evaluated 2 hours after a single intraperitoneal injection at 2 mg/Kg. INX201J, INX231J, INX234J, and INX240J effects were evaluated 4 days (-4) and 7 days (-7) after a single intraperitoneal injection at 10 mg/Kg. Cell supernatants were collected at 2 hours. TNFα and IL-6 were measured using ELISA (n=4 mice/group; conventional one-way ANOVA compared to PBS only group). Includes the results of experiments evaluating the efficacy of exemplary ADC conjugates according to the invention, namely INX231J, INX234J, and INX240J. Results show that INX231J, INX234J, and INX240 J ADCs are equally effective in preventing ex vivo induction of TNFα and IL-6 during PRM. Dex effects were evaluated 2 hours after a single intraperitoneal injection at 2 mg/Kg. INX231J, INX234J, and INX240J effects at 7 days after a single intraperitoneal injection at 10, 3, or 1 mg/Kg (equivalent to 0.2, 0.06, and 0.02 mg/Kg GC payload). evaluated. Cell supernatants were collected at 2 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group, except PBS group n=1 for technical reasons; compared with PBS only group) standard one-way ANOVA). Includes experiments showing that the efficacy of INX201J, INX201P, INX231J, INX234J, and INX240J ADCs are equivalent in preventing ex vivo induction of TNFα and IL-6 during PRM. INX201J, INX201P, INX231J, INX234J, INX240J and Dex effects were evaluated 7 days after a single intraperitoneal injection. ADC was administered at 10 mg/Kg (GC payload of 0.2 mg/Kg) and Dex at 2 mg/Kg. Cell supernatants were collected at 2 hours. ELISA was used to measure TNFα and IL-6 (n = 4 mice/group, excluding PBS and Dex groups which included n = 3 for technical reasons; normal mice compared to PBS only group). direction ANOVA). INX201J, INX231P, INX234P, and INX240P ADCs are equally effective in preventing ex vivo induction of TNFα during PRM. ADC efficacy was evaluated 7 days after a single intraperitoneal injection. ADC was administered at 10 mg/Kg (GC payload of 0.2 mg/Kg). Cell supernatants were collected at 2 hours. TNFα was measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). INX231P, INX231R, INX233P and INX234P are equally effective in preventing ex vivo induction of TNFα and IL-6 during PRM. ADC efficacy was evaluated 7 days after a single intraperitoneal injection. ADC was administered at 10 mg/Kg (GC payload of 0.2 mg/Kg). Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). Efficacy evaluation of GC linker payloads INX R, INX O, INX S, INX V and INX W versus INX P conjugated to INX231 in preventing ex vivo induction of TNFα and IL-6 in PRM. ADC efficacy was evaluated 7 days after a single intraperitoneal injection. ADC was administered at 0.2 mg/Kg GC payload. Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). Efficacy evaluation of GC payloads INX231S, INX231V, and INX231W versus INX231P in inducing FKBP5 transcription in PRM. ADC efficacy was evaluated on days 1, 7, and 14 after a single intraperitoneal injection. ADC was administered at 0.2 mg/Kg GC payload. FKBP5 expression was measured by quantitative real-time PCR and presented as Log2 fold change versus PBS control group (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). Efficacy evaluation of GC payloads INX231S, INX231V, and INX231W versus INX231P in preventing ex vivo induction of TNFα and IL-6 during PRM. ADC efficacy was evaluated on days 1, 7, and 14 after a single intraperitoneal injection. ADC was administered at 0.2 mg/Kg GC payload. Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). Efficacy evaluation of GC payloads INX234A3, INX234A4, INX234T, INX201L and INX231S in inducing FKBP5 transcription in peritoneal resident macrophages (top row) and splenocytes (bottom row). ADC efficacy was evaluated on days 1, 7, and 14 after a single intraperitoneal injection. ADC was administered at 0.2 mg/Kg GC payload. FKBP5 expression was measured by quantitative real-time PCR and presented as Log2 fold change versus PBS control group (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). Efficacy evaluation of GC payloads INX234A3, INX234A4, INX234T, INX201L, and INX231S in preventing ex vivo induction of TNFα and IL-6 during PRM. ADC efficacy was evaluated on days 1, 7, and 14 after a single intraperitoneal injection. ADC was administered at 0.2 mg/Kg GC payload. Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). Evaluation of the efficacy of GC payloads INX234V, INX234A5 and INX234A11 in inducing FKBP5 transcription in peritoneal resident macrophages. ADC efficacy was evaluated on days 7 and 14 after a single intraperitoneal injection. ADC was administered at 0.2 mg/Kg GC payload. FKBP5 expression was measured by quantitative real-time PCR and presented as Log2 fold change versus PBS control group (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). Efficacy evaluation of GC payloads INX234V, INX234A5, and INX234A11 in preventing ex vivo induction of TNFα and IL-6 during PRM. Fourteen days after a single intraperitoneal injection, ADC efficacy was assessed and ADC TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; Conventional one-way ANOVA compared to PBS only group, SEM). Evaluation of the efficacy of GC payloads INX234V, INX231A7, INX231A12, and INX231A23 in inducing FKBP5 transcription in peritoneal resident macrophages. ADC efficacy was evaluated on days 7 and 21 after a single intraperitoneal injection. ADCs were administered with a GC payload of 0.2 mg/Kg, except INX231A7, which was administered with a payload of 0.08 mg/Kg. FKBP5 expression was measured by quantitative real-time PCR and presented as Log2 fold change versus PBS control group (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). Efficacy evaluation of GC payloads INX234V, INX231A7, INX231A12, and INX231A23 in preventing ex vivo induction of TNFα and IL-6 during PRM. ADC efficacy was evaluated on days 7, 14, and 21 after a single intraperitoneal injection. ADC was administered at 0.2 mg/Kg GC payload. Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). Changes in IL-12p40 at 2 hours (left) and 4 hours (right) after LPS in peripheral blood. Plasma concentrations measured using mouse multiplex, administration: Dex (squares) administered 2 hours before LPS stimulation at 0.02, 0.2, 2 and 5 mg/Kg, INX201J (circles) , administered 2 hours or 17 hours before LPS injection at 10 mg/Kg to provide 0.2 mg/Kg of GC. The PBS only group (gray filled triangles) represents baseline cytokine levels in the absence of stimulation. PBS+LPS (filled triangles) (SEM; n=5/group, unless technical failures are excluded from analysis; conventional one-way ANOVA compared to PBS+LPS group). Cytokine changes 2 hours after LPS in peripheral blood. Plasma concentrations measured using mouse 5plex, administration: Dex at 0.002, 0.02, 0.2, 2 mg/Kg 2 hours before LPS stimulation (squares) or LPS at 2 mg/Kg. INX201J (circle) was administered 17 hours before LPS injection in GC payloads of 0.02, 0.06, and 0.2 mg/Kg. The PBS only group (gray filled triangles) represents baseline cytokine levels in the absence of stimulation. PBS+LPS (filled triangles) (SEM; n=5/group, unless technical failures are excluded from analysis; conventional one-way ANOVA compared to PBS+LPS group). Changes in TNFα 2 hours after LPS in peripheral blood. TNFα plasma concentrations were measured using ELISA. Administration: Dex was administered 2 hours before LPS stimulation at 0.2 and 2 mg/Kg (squares), INX201J (circles) at 0.06 and 0.2 mg/Kg GC payload 17 hours after LPS injection. administered before. The PBS group (filled triangles) received PBS 2 hours before LPS. The IgG1siJ (G1siJ) group (triangles) received human IgG1 silently conjugated to GC in a payload of 0.2 mg/Kg 17 hours before LPS. (SEM; n=5/group, except when technical failures are excluded from analysis; normal one-way ANOVA compared to PBS group). Changes in TNFα 2 hours after LPS in peripheral blood. TNFα plasma concentrations were measured by ELISA. Administration: Dex was administered 2 hours before LPS stimulation at 0.2 and 2 mg/Kg (squares), INX201J (circles) and INX201N (inverted triangles) at 0.2 mg/Kg GC payload before LPS injection. Administered 17 hours prior. The PBS group received PBS 2 hours before LPS (filled triangles). (SEM; n=5/group, except when technical failures are excluded from analysis; normal one-way ANOVA compared to PBS group). Changes in TNFα (left) and IL-12p40 (right) 2 hours after LPS in peripheral blood are shown. Cytokine plasma concentrations were measured by ELISA. Administration: PBS (filled circles), INX201J (squares), INX231J (triangles), INX234J (diamonds), and INX201P (inverted triangles) were administered 17 hours before LPS injection in a GC payload of 0.2 mg/Kg. (SEM; n=5/group; conventional one-way ANOVA compared to PBS group). Changes in TNFα (left) and IL-12p40 (right) 2 hours after LPS in peripheral blood are shown. Cytokine plasma concentrations were measured by ELISA. Administration: PBS (filled triangles), INX201J (circles), INX201O (squares), and INX201P (diamonds) were administered 17 hours before LPS injection in a GC payload of 0.2 mg/Kg (SEM; technical failure n = 5/group; normal one-way ANOVA compared to PBS group). Changes in TNFα (right) and IL-12p40 (left) 2 hours after LPS in peripheral blood are shown. Cytokine plasma concentrations were measured by ELISA. Administration: PBS, INX201J (circles), INX201O (squares), and INX201P (diamonds) were administered 17 hours before LPS injection in 0.2 mg/Kg GC payload (SEM; technical failures were excluded from analysis. n = 5/group; normal one-way ANOVA compared to PBS group (filled triangles). Changes in TNFα (right) and IL-12p40 (left) 2 hours after LPS in peripheral blood are shown. Cytokine plasma concentrations were measured by ELISA. All ADCs and PBS were administered 20 hours before LPS injection at 0.2 mg/Kg GC payload (INX231P (squares), INX231R (triangles), INX233P (diamonds)) (SEM; technical failure prevented analysis n = 5/group, except where excluded; normal one-way ANOVA compared to PBS group (filled circles). Changes in TNFα (right) and IL-12p40 (left) 2 hours after LPS in peripheral blood are shown. Cytokine plasma concentrations were measured by ELISA. All ADCs and PBS were administered 20 hours before LPS injection at GC payload of 0.2 mg/Kg (INX231P (filled squares), INX231R (filled triangles), INX201O (filled diamonds), INX231S (circles). ), INX231V (squares), INX231W (triangles)) (SEM; n = 4/group, except for INX231S where two technical failures are excluded from the analysis; normal unidirectional compared to the PBS group (filled circles) ANOVA showed non-significant data). FIG. 4 shows FKBP5 transcriptional activation after ADC treatment in peritoneal residents 4 days after ADC treatment. ADCs were injected intraperitoneally on day 0, each delivering a GC payload of 0.2 mg/Kg. PRMs were isolated on day 3. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus PBS control group (SEM, conventional one-way ANOVA compared to PBS group, n=4). Changes in TNFα (right) and IL-12p40 (left) 2 hours after LPS in peripheral blood. Cytokine plasma concentrations were measured by ELISA. All ADCs and PBS were administered 20 hours before LPS injection at GC payload of 0.2 mg/Kg (SEM; except INX234P, INX234A4, and INX234T where one technical failure was recorded per group, n= 5/group; conventional one-way ANOVA compared to PBS group). Changes in TNFα (right) and IL-12p40 (left) 2 hours after LPS in peripheral blood. Cytokine plasma concentrations were measured by ELISA. All ADCs and PBS were administered 20 hours before LPS injection at GC payload of 0.2 mg/Kg (INX234V (filled squares), INX234A5 (filled triangles), INX234A11 (filled diamonds)) (SEM; n=5/group; conventional one-way ANOVA compared to PBS (filled circles) group). Changes in TNFα (right) and IL-12p40 (left) 2 hours after LPS in peripheral blood. Cytokine plasma concentrations were measured by ELISA. All ADCs and PBS were administered 20 hours before LPS injection at a GC payload of 0.2 mg/Kg, except INX231A7, which was administered at a payload of 0.08 mg/Kg (INX234V (filled squares), INX231A7 ( (filled triangles), INX231A12 (filled diamonds), INX231A23 (circles), INX234A1 (squares), INX234A13 (triangles)) (SEM; n=5/group; conventional one-way ANOVA compared to PBS group). Changes in TNFα (right) and IL-12p40 (left) 2 hours after LPS in peripheral blood. Cytokine plasma concentrations were measured by ELISA. All ADCs and PBS were administered 20 hours before LPS injection at GC payload of 0.2 mg/Kg (SEM; PBS, INX234P, one sample that needed to be censored within the INX201V group, and technical Excluding 2 samples in the INX234A9 group for reasons, n=5/group; normal one-way ANOVA compared to PBS group). Contains the results of experiments detecting VISTA expression on different cells. As shown therein, VISTA is highly expressed in liver endothelial cells. CD45-CD31+ non-immune endothelial cells isolated from hVISTA knock-in mouse livers and stained with anti-human VISTA (red line, shifted to the right) or unstained (solid grey). Contains the results of experiments detecting FKBP5 transcriptional activation after INX201J injection in adrenal gland, brain, liver, and spleen. 20 after a single intraperitoneal injection at 0.3, 3, 10 mg/Kg (delivering payloads of 0.006, 0.06, and 0.2 mg/Kg, respectively) as shown therein. At the same time, the INX201J effect was measured. Dex effects were measured 2 hours after a single intraperitoneal injection at 0.2 or 2 mg/Kg. FKBP5 transcript levels were measured by real-time PCR and presented as the Log2 fold change versus the mean of the PBS control group (n=4 mice/group, conventional one-way ANOVA compared to the PBS only group). INX-SM-3, INX-SM-4, and INX-SM-1 inhibit the production of IL-1β (left) and IL-6 (right). Cytokine levels were measured over 24 hours for human PBMCS incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-1 nM), with <1 nM no treatment control plotted on the x-axis on a logarithmic scale. , n=1 donor and standard deviations were plotted from technical duplicates. INX-SM-1, INX-SM-3, INX-SM-4 and INX-SM-6 inhibit IL-1β production. Cytokine levels were measured over 24 hours for human PBMCS incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-1 nM), with <1 nM no treatment control plotted on the x-axis on a logarithmic scale. , n=1 donor and standard deviations were plotted from technical duplicates. INX-SM-9, INX-SM-31, and INX-SM-35 inhibit the production of IL-1β (top) and IL-6 (bottom). Cytokine levels were measured at 24 hours for human PBMCS incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.2 nM), with <0.2 nM marked as treatment control on the x-axis on a logarithmic scale. are not plotted, n=2 donors, and a representative donor is shown. Standard deviation plotted from technical duplicates. INX-SM-32 inhibits the production of IL-1β (top) and IL-6 (bottom). Cytokine levels were measured over 24 hours for human PBMCS incubated with 1 ng/mL LPS and serial dilutions of steroid payload (500-1 nM), with <1 nM no treatment control plotted on the x-axis on a logarithmic scale. , n=2 donors. Representative donors are shown. Standard deviation plotted from technical duplicates. INX-SM-10 shows robust inhibition in IL-1β (top) and IL-6 (bottom) production. INX-SM-33 demonstrated modest inhibition of cytokine production. Cytokine levels were measured at 24 hours for human PBMCS incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.5 nM), with <0.5 nM marked as treatment control on the x-axis on a logarithmic scale. is not plotted, n=1 donor, and standard deviations are plotted from technical duplicates. INX-SM-2 and INX-SM-7 show inhibition at IL-1β. Cytokine levels were measured at 24 hours for human PBMCS incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.16 nM), with <0.16 nM marked as treatment control on the x-axis on a logarithmic scale. is not plotted, n=1, and standard deviations are plotted from technical duplicates. We show that halogenation at both C6 and C9, rather than C9 alone, provides increased potency. For human PBMCS incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.16 nM), mean cytokine levels were measured over 24 hours, with <0.16 nM treated on the x-axis on a logarithmic scale. No controls were plotted, n=1, and standard deviations were plotted from technical duplicates. INX P-conjugated antibodies directed against other surface targets retain the anti-inflammatory effects of the anti-VISTA conjugate. Mean cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of glucocorticoid conjugate (conjugate payload from 8,000 to 0.26 nM). n=1, mean technical overlap. INX-SM-43 shows moderate inhibition of huIL1-β. Mean cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of glucocorticoid payload (100-0.032 nM). n=1, mean technical overlap. INX-SM-44 shows moderate inhibition of huIL1-β. Mean cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of glucocorticoid payload (1000-0.32 nM). n=1, mean technical overlap. INX-SM-25 and INX-SM-3 show robust inhibition in IL-1β production. INX-SM-45 and INX-SM-46 demonstrated more modest inhibition of cytokine production. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.5 nM), with <0.5 nM marked as treatment control on the x-axis on a logarithmic scale. is not plotted, n=1 donor, and the mean of technical duplicates is plotted. Dramatically increased efficacy of INX231V compared to INX231P and INX231J. A. for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM). huIL-1β B. Mean cytokine levels of huIL-6 were measured at 24 hours, <0.16 nM, no treatment control plotted on the x-axis on a logarithmic scale, n=1, and standard deviations were plotted from technical duplicates. Values above the ULOQ (12,500 pg/mL for IL-1b and 150,000 pg/mL for IL-6) were plotted as inserted values. INX231V has substantial potency and INX231P has modest potency compared to INX231J. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted. Improved efficacy of INX231S, INX234T and INX234A3 compared to INX231J. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted. INX201 may lead to improved early efficacy of the conjugate versus INX231. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted. Analogs of INX V are more potent compared to INX231J. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted. INX-SM-36, INX-SM-32 (top) and INX-SM-3, INX-SM-J2 (bottom) inhibit IL-1β. INX-SM-32 and INX-SM-36 inhibit IL-1β with similar potency as dexamethasone. INX-SM-3 and INX-SM-J2 have similar efficacy to budesonide. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.5 nM), with <0.5 nM marked as treatment control on the x-axis on a logarithmic scale. is not plotted, n=1 donor, and the mean of technical duplicates is plotted. INX-SM-32, INX-J2, and INX-SM-3 induce similar inhibition of IL-1β, and INX-SM-37 weakly inhibits IL-1β. INX-SM-32, INX-J2 and INX-SM-3 inhibit IL-1β with similar efficacy. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.15 nM), with <0.5 nM marked as treatment control on the x-axis on a logarithmic scale. is not plotted, n=1 donor, and the mean of technical duplicates is plotted. INX231V is substantially more potent than other INX231/INX234 conjugates. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted. Phosphorylated and halogenated analogs of INX V are more potent compared to INX J. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted. INX-SM-14, INX-SM-15, and INX-J2 have similar inhibition of IL-1β (top) and IL-6 (bottom). INX-SM-17 weakly inhibits IL-1β but not IL-6. INX-SM-14, INX-SM-15 and INX-J2 inhibit IL-1β and IL-6 with similar efficacy. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.15 nM), with treatment control on the x-axis of a logarithmic scale at 0.01 nM. Not plotted, n=1 donor, mean of technical duplicates plotted. INX-SM-40 and INX-SM-34 weakly inhibit IL-1β (top) and IL-6 (bottom) compared to INX-J2. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.15 nM), with treatment control on the x-axis of a logarithmic scale at 0.01 nM. Not plotted, n=1 donor, mean of technical duplicates plotted. INX-SM-49 and INX-SM-47 weakly inhibit IL-1β (top) and IL-6 (bottom) compared to INX-J2. Cytokine levels (IL1-β and IL-6) were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.15 nM), logarithmic at 0.01 nM. No treatment control was plotted on the x-axis of the scale, n=1 donor, and the mean of technical duplicates was plotted. INX231A9 and INX201V show improved efficacy compared to INX234J and INX201J in reducing IL-1β production. Cytokine levels were measured at 24 hours for human PBMCs incubated with serial dilutions of anti-VISTA conjugate to 1 ng/mL LPS and conjugate payload concentrations (1000 to 0.15 nM), and 0.1 nM on a logarithmic scale. No treatment control was plotted on the x-axis, n=1 donor, and the mean of technical duplicates was plotted. INX V and INX A23 at equivalent or reduced DAR show improved efficacy compared to INX J in reducing IL-1β production. Cytokine levels were measured at 24 hours for human PBMCs incubated with serial dilutions of anti-VISTA conjugate to 1 ng/mL LPS and total ADC concentrations (20 to 0.003 μg/mL) and 0.001 μg/mL. No treatment controls were plotted on the log scale x-axis, n=1 donor, and the mean of technical duplicates was plotted. INX V conjugate has improved potency compared to INX J conjugate in affecting IL-1β even with reduced DAR. Cytokine levels were measured at 24 hours on human PBMCs incubated with serial dilutions of anti-VISTA conjugate to 1 ng/mL LPS and conjugate payload concentrations (20 to 0.003 μg/mL) and 0.001 μg/mL. No treatment control was plotted on the x-axis on a logarithmic scale, n=1 donor, and the mean of technical duplicates was plotted. Contains the results of experiments comparing the PK properties of the exemplary inventive antibody INX200 versus human IgG1. Plasma concentrations of antibodies at annotated time points in hVISTA KI mice (SD; n=5/group) as indicated therein. Contains the results of experiments comparing the PK properties of 767-IgG1.3 versus human IgG1. Plasma concentrations of antibodies at annotated time points in hVISTA KI mice (SD; n=5/group) as indicated therein. Includes the results of experiments comparing the PK values of other exemplary anti-VISTA antibodies according to the invention, namely INX231, INX234, INX237, and INX240. Plasma concentrations of antibodies at annotated time points in hVISTA KI mice (SD; n=5/group) as indicated therein. The left graph shows the y-axis and x-axis at Log10, while for the right graph only the y-axis is at Log10. Contains the results of experiments comparing the PK values of exemplary anti-VISTA antibodies according to the invention, namely INX901, INX904, INX907, and INX908. Plasma concentrations of antibodies (SD; n=5/group) at annotated time points in hVISTA KI mice. Contains the results of experiments comparing the PK values of different ADCs according to the invention, namely INX201J, INX231J, INX234J, and INX240J. Plasma concentrations of antibodies at annotated time points (SD; n=4/group) in hVISTA KI mice. Includes results of experiments assaying the effects of long-term treatment with exemplary VISTA Ab ADC conjugate INX201J and dexamethasone on corticosterone levels. The figure shows changes in plasma corticosterone levels. (SEM, one-way ANOVA, excluding PBS control group in right graph with n=6, n=8). The number of Ag-specific CD8 T cells from peripheral blood on day 6 after immunization in Experiment 1 of Example 12 is shown. (SEM, one-way ANOVA, n=5). The number of Ag-specific CD8 T cells from peripheral blood on day 6 after immunization in Experiment 2 of Example 12 is shown. The graph on the left shows the PBS control group with all samples, the graph on the right shows the PBS control group with one outlier removed (SEM, one-way ANOVA, n=5 excluding naive, one Samples were excluded in the group containing Dex at 0.2 mg/Kg as immunization failures). The number of Ag-specific CD8 T cells from peripheral blood on day 6 after immunization in Experiment 3 of Example 12 is shown. In this experiment, several samples had to be excluded due to technical issues during processing. PBS group n=3, Dex at 2 mg/Kg n=2, Dex at 0.2 mg/Kg n=3, INX201J D-1 n=5, INX201J D-7 n=2, INX231J D-7 n=3, INX234 J D-7 n=5, INX240 J D-7 n=4 (SEM, one-way ANOVA, D=day). The number of Ag-specific CD8 T cells from peripheral blood on day 6 after immunization in Experiment 3 of Example 12 is shown. Due to technical reasons, two samples were excluded in the PBS, INX231P and INX234P groups, n=5 for all other groups (SEM, one-way ANOVA). Figure 2 shows changes in absolute cell numbers in peripheral blood in two experimental schedules. OVA challenge on days 14-18 and 21-25 (SEM, one-way ANOVA, n=10 excluding naive including n=5). Figure 2 shows changes in immunoglobulin production in peripheral blood in two experimental schedules. OVA challenge on days 14-18 (Part 1) and days 21-25 (Part 2) (SEM, one-way ANOVA, n=10 excluding naive including n=5). Figure 2 shows changes in immune infiltration in BAL in two experimental schedules. OVA challenge on days 14-18 (Part 1) and days 21-25 (Part 2), A) Changes in bone marrow infiltration, B) Changes in lymphocyte infiltration (SEM, one-way ANOVA, censored by control group) (n=10, n=5 for the naive group), including 3 in both the Dex and INX201J groups. Figure 2 shows changes in cytokine levels in BAL in two experimental schedules. OVA challenge on days 14-18 (Part 1) and days 21-25 (Part 2) (SEM, one-way ANOVA, 2 samples censored in control group, 3 in both Dex and INX201J groups) n = 10, n = 5 for the naive group). Figure 2 shows lung disease scoring for part 1 of the study. (SEM, one-way ANOVA, n=10, excluding naive group n=5). FKBP5 transcriptional activation after INX231J injection in spleen (left) and blood (right) cells is shown. INX231J effects and hIgG1siJ (gray) were measured 20 hours after a single intravenous injection at 5 mg/Kg (delivering a payload of 0.1 mg/Kg). Dex effects were measured 2 hours after a single intraperitoneal injection at 2 mg/Kg. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus the mean of the PBS control group. (n=4 mice/group, conventional one-way ANOVA compared to PBS only group. Figure 2 shows FKBP5 transcriptional activation after INX231P injection in C57Bl/6 mice. INX231P effects were measured 20 hours after a single intravenous injection at 10 mg/Kg (delivering a payload of 0.2 mg/Kg). Dex effects were measured 2 hours after a single intraperitoneal injection at 2 mg/Kg. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus the mean of the PBS control group. (n=4 mice/group, normal one-way ANOVA compared to PBS only group). Contains experimental results showing FKBP5 transcriptional activation following INX231P injection in C57Bl/6 or hVISTA KI mice. INX231P effects were measured 20 hours after a single intravenous injection at 10 mg/Kg (delivering a payload of 0.2 mg/Kg). Dex effects were measured 2 hours after a single intraperitoneal injection at 2 mg/Kg. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus the mean of the PBS control group. (n=4 mice/group, normal one-way ANOVA compared to PBS only group). Includes experimental results showing that in vivo Dex treatment causes a reduction in ex vivo monocyte inflammatory responses to LPS. Mice were injected intraperitoneally with PBS or Dex at 2 mg/Kg or 0.2 mg/Kg. Two hours later, splenic monocytes were isolated, cultured, and stimulated with LPS at 0, 10, and 100 ng/ml. 24-hour supernatants were analyzed on a Luminex 32-plex (n=5 mice/group, but samples 1, 2, 3, and 4, 5 were pooled into two samples). Includes experimental results showing that in vivo treatment with INX231P affects ex vivo monocyte inflammatory responses to LPS. Mice were injected intraperitoneally with PBS or Dex at 2 mg/Kg 2 hours, 2 days, or 6 days before cell isolation and at 10 mg/Kg 1 day, 3 days, and 7 days before cell isolation. INX231P and INX901 were injected intravenously. After isolation, splenic monocytes were cultured and subjected to LPS stimulation at 0 or 10 ng/ml (only 10 ng/ml is shown). 24-hour supernatants were analyzed by ELISA (n=4 mice/group, one-way ANOVA comparing to PBS-treated group was performed only on day 1 (D1) samples). Includes experimental results showing that in vivo treatment with INX231P affects ex vivo monocyte inflammatory responses to LPS. Mice were injected intraperitoneally with PBS or Dex at 2 mg/Kg 2 hours before cell isolation and intravenously with INX231P and INX901 at 10 mg/Kg 24 hours before cell isolation. Splenic monocytes were cultured and stimulated with LPS at 10 and 100 ng/ml. 24 hour supernatants were analyzed by ELISA (n=4 mice/group, separate routine one-way ANOVA compared to PBS treated group for each LPS dose). FKBP5 transcriptional activation in B cells or monocytes is shown. Cells were treated with 20 nM free J payload, or payload conjugated to equimolar amounts of INX201 (INX201J) or isotype control (huIgG1si J). Transcript levels were analyzed as technical duplicates. FKBP5 transcriptional activation in monocytes is shown. Cells were treated with increasing amounts of INX201J [0-100 nM payload]). 0 payload represents treatment with unconjugated INX201 antibody alone at the same amount of antibody as the 100 nM payload INX201J dose. Transcript levels were analyzed as technical duplicates. FKBP induction in T regs from two donors treated with 20 nM INX-SM-3 (free payload) or molar payload equivalents of INX231P (conjugated payload) is shown. Samples were generated and analyzed as single units. The purity of isolated Tregs was ≧75% as assessed by flow cytometry. Figure 3 shows FKBP5 induction in T reg from one donor treated with 20 nM payload equivalent of INX201J compared to 20 nM payload equivalent of huIgG1si J. Samples were analyzed as technical duplicates. The purity of isolated Tregs was ≧75% as assessed by flow cytometry. TPM<10 represents (minimal/no expression "-"), TPM 10 to 100 represents (low/medium expression "+"), TPM>100 represents (high expression "++"), Summarizes the reported consensus RNA expression levels by different area cells for VISTA and other ADC targets (CD40, TNFα, CD74, CD163 (PRLR) based on reported “transcripts per million” (TPM) . Summarizing the quantification of antigen density of VISTA, CD74, CD163, and mTNFα on identified cell populations, A) monocytes express VISTA, CD74, and CD163; B) B cells express CD74. and C) CD4+ T cells, D) CD4+ T reg, E) CD8+ T cells express VISTA (mean±SD, n=5 donors). Summarizing the quantification of antigen density of VISTA, CD74, CD163, and mTNFα on identified cell populations in human blood, A) monocytes express VISTA, CD74, and CD163; B) B cells express VISTA, CD74, and CD163; , CD74, C) Neutrophils express VISTA, D) CD4+ T cells, E) CD4+ T reg, F) CD8+ T cells express VISTA (mean±SD, n=3). Contains data showing that steroid-responsive genes in bone are significantly affected by free Dex, while VISTA ADC (INX231P) has a limited effect. In the figure, Fkpb5 is shown on the left, RANKL is shown on the center left, ddit4 is shown on the center right, and Fam107a is shown on the right end. In the experiment, INX231P effects were measured 20 hours after a single intraperitoneal injection at 10 mg/Kg (delivering a payload of 0.2 mg/Kg). Dex effects were measured 2 hours after a single intraperitoneal injection at 2 mg/Kg. Gene transcript levels were measured by RNAseq and presented as normalized counts (n=5 mice/group, conventional one-way ANOVA compared to PBS only group). Contains the results of experiments comparing the effects of VISTA ADC (INX234P) compared to vehicle controls on steroid-responsive gene expression in peripheral blood cells of cynomolgus monkeys. In these experiments, INX234P effects were measured 24 hours after a single intravenous dose at 10 mg/Kg (delivering a payload of 0.2 mg/Kg). Gene transcript levels were measured by RNAseq and presented as normalized counts (n=2 vehicles, n=6 ADCs/group, unpaired T-test vs. vehicle). Contains the results of experiments evaluating the effects of VISTA ADC (INX234P) on specific non-target cells. As shown by the data, INX234P had limited or no effect on FKBP5 in white fat, brain, and bone. In the experiment, INX234P effects were measured 24 hours or D8 (vehicle) after a single intravenous dose at 10 mg/Kg (delivering a payload of 0.2 mg/Kg). Gene transcript levels were measured by RNAseq and presented as normalized counts. (n=2 vehicle, n=3 ADC/group for tissue, unpaired T-test vs. vehicle-INX234P was not significant). Contains the results of experiments evaluating the effects of ADCs on steroid-responsive genes. The data show some residual Dex effect at 24 hours in white adipose tissue, with ADC gene expression similar to vehicle control. The effects of free Dex (2 mg/Kg) and INX234P (10 mg/Kg, delivering a payload of 0.2 mg/Kg) were determined 24 hours or day 8 (vehicle) after a single intravenous administration. Gene transcript levels were measured by RNAseq and presented as normalized counts. (n=2 vehicles, n=2 dexamethasone, n=3 ADC/group for tissues, unpaired T-test vs. vehicle). Includes experimental results showing that high accumulation of active payload (INX-SM-3) at 24 hours in monkeys treated with INX234P correlates with VISTA expressing tissues. Panel (A) shows the release measured 24 hours after a single intravenous dose of either INX234P (10 mg/kg, delivering a payload of 0.2 mg/Kg) or free dexamethasone (2 mg/kg). Panel (B) shows the cysteine-modified linker/payload (INXP-cys) and Panel (C) shows dexamethasone. Panel (D) shows that on day 8, the released payload (INX-SM-3) persisted in the VISTA-expressing tissues of monkeys treated with INX234P. Accumulated compound levels were measured by LC-MS/MS and presented as ng of compound/g of tissue (n = 3 ADCs/group for INX234P excluding bone marrow on day 8, due to limited samples. n=2, n=2 Dex) (Duod=duodenum). Expression of VISTA is compared to other proteins on activated immune cells (monocytes), particularly those targeted by other steroid ADCs. In experiments, human whole blood from healthy donors was activated with LPS (100 μL per well in a U-bottom 96-well plate, 1 μg/mL LPS, 2 hours at 37° C.). Cell surface protein expression levels on activated immune (monocyte) cells were assessed by flow cytometry. Directly conjugated antibodies used for staining in these experiments include anti-VISTA clone GG8, CD163 clone GHI/61, CD74 clone 332516, and mTNFα clone mAb11. As shown, the VISTA expression pattern was similar to the expression levels observed on non-activated cells, while other protein expression levels were low. Specifically, mTNFα MFI was only slightly higher than the FMO (fluorescence minus one) control. Contains the results of experiments evaluating the PK of an ADC according to the invention, namely INX234P, in cynomolgus monkeys. INX234P was administered intravenously at 15 mg/Kg. Animals were bled at the indicated time points, serum was isolated, and antibody levels were measured (n=4 cynomolgus monkeys, SEM). Figure 2 shows hematological changes induced by a single dose of INX234P. In these experiments, INX234P was administered intravenously at 15 mg/Kg. Animals were bled at the indicated time points, blood smears were performed, and different cell populations were counted (n=4 cynomolgus monkeys, SEM) (WBC=white blood cells, NEUT=neutrophils, LYMPH=lymphocytes, MONO=monocytes). globules, EOS = eosinophils, BASO = basophils, RBC = red blood cells, Retic = reticulocytes). Contains the results of experiments detecting cortisol changes induced by a single dose of INX234P in individual animals. In these experiments, INX234P was administered intravenously at 15 mg/Kg. Animals were bled at the indicated time points, serum was isolated, and cortisol levels were measured by ELISA (n=4 cynomolgus monkeys). Contains the results of experiments detecting the PK of the linker payload (P-cys) and released payload (SM3) in PBL. INX234P was administered intravenously at 15 mg/Kg. Animals were bled at the indicated time points and PBLs were isolated and analyzed by MS (n=4 cynomolgus monkeys, SEM). Contains the results of a PK assay that detected the PK of linker payload (P-cys) and released payload (SM3) in serum. INX234P was administered intravenously at 15 mg/Kg. Animals were bled at the indicated time points and serum was isolated and analyzed by MS (n=4 cynomolgus monkeys, SEM). Contains the results of experiments showing that steroid-responsive genes in PBL are upregulated by INX234P. In these experiments, INX234P was administered intravenously at 15 mg/Kg. Animals are bled at the indicated time points, PBLs are isolated, subjected to RNA isolation, and gene transcript levels are measured by RNAseq and presented as fold change versus pre-bleeding (n=4 cynomolgus monkeys). Includes experiments showing the effects of INX201J, INX231P, and INX231V on FKBP5 induction in human PBMCs for up to 4 hours. In experiments, n=1 donor, FKBP5 induction measured by RT PCR compared to GAPDH was detected in human PBMCs incubated with 1 μM conjugate payload for 4 hours. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Includes trade names and chemical names and structures of exemplary glucocorticoid agonist compounds and glucocorticoid agonist-linker compounds of Formulas I, II, and III. Contains results from GVHD experiments showing that INX234P treatment reduces human PBMC proliferation. In these experiments, peripheral blood was collected on day 21 and human CD45-positive cells were quantified by flow cytometry. Mice were dosed once a week from day 0 to day 34 (SEM, n=8/group) (dosing at 10 mg/Kg, INX234P provided 0.2 mg/kg of INX P linker payload). Contains GVHD experimental results showing that INX234P treatment improves mouse survival. Mice were dosed intraperitoneally at 10 mg/Kg (or 0.2 mg/Kg INX P linker payload) once a week from day 0 to day 34. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve, with the upper ( The gray bar below (bottom graph) or below (top graph) indicates the treatment period (SEM, n=8/group). Includes GVHD experiments showing that INX234V/P treatment reduces human T cell proliferation. Peripheral blood was collected weekly starting from day 15 post-transplant, and human CD45+CD3+ positive cells were quantified by flow cytometry. Mice were dosed once a week from day 0 (SEM, n=8/group) (dosing at 10 mg/Kg, INX234V and INX234P received 0.2 mg/kg of INX V or INX P linker payload, respectively). provided). Contains data obtained in a GVHD model showing that INX234V/P treatment improves mouse survival. Mice were dosed intraperitoneally at 10 mg/Kg (or 0.2 mg/Kg INX V or INX P linker payload) once a week starting on day 0. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve. Contains data obtained in a colitis model showing that INX234P treatment improves mouse survival. In the experiment, mice were given both INX234P and INX234 at 10 mg/Kg (and 0.2 mg/Kg INX P linker payload if applicable) intraperitoneally once a week from day 0 to day 61. was administered. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve, with the upper ( The gray bar below (bottom graph) or below (top graph) indicates the treatment period (SEM, excluding INX234P group containing n=9, n=10/group). Contains data obtained in a colitis model showing that high-dose dexamethasone treatment improves mouse survival. In the experiment, mice were dosed intraperitoneally at 2 (high) or 0.2 (low) mg/Kg once a week from day 0 to day 61. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve, with the upper ( The gray bar below (bottom graph) or below (top graph) indicates the treatment period (SEM, n=10/group). Contains data obtained in a colitis model showing that INX234V treatment improves mouse survival. In the experiment, mice were given 10 mg/Kg (and 0.2 mg/Kg INX V linker payload if applicable) for both INX234V and INX234 intraperitoneally once a week from day 21 to day 80. was administered. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve, with the upper ( The gray bar below (bottom graph) or below (top graph) indicates the treatment period (SEM, excluding INX234 treatment group where n=5, n=10/group). Contains data obtained in a colitis model showing that low-dose dexamethasone treatment improves mouse survival. In the experiment, mice were dosed intraperitoneally at 2 (high) or 0.2 (low) mg/Kg once a week from day 21 to day 80. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve, with the upper ( The gray bar below (bottom graph) or below (top graph) indicates the treatment period (SEM, n=10/group). Contains data obtained in a colitis model showing that INX234V treatment prevents T cell proliferation and activation. In the experiment, mice were given 10 mg/Kg for INX234V or INX234 (and 0.2 mg/Kg V-linker payload, if applicable) and 2 (high) or Dex was administered intraperitoneally at 0.2 (low) mg/Kg. The top left graph shows the number of naïve T cells from blood for Dex in INX234V and INX234 vs. PBS on the left and high and low doses vs. PBS on the right, and the bottom left and right graphs show the number of CD45 ^RB+ CD4 for the same group. The frequency of ⁺ T cells is shown, and the gray bar above indicates the treatment period (SEM, excluding INX234 treatment group where n=5, n=10/group).

Described herein are novel glucocorticosteroids, glucocorticosteroid linkers, comprising antibodies or antibody fragments that bind to antigens expressed on immune cells, typically human immune cells; and antibody drug conjugates (ADCs) are provided. In some embodiments, the ADC is an anti-human VISTA (V region immunoglobulin-containing suppressor of T cell activation (1)) antibody or anti-VISTA antigen-binding antibody fragment, e.g., a human VISTA knockin with a short serum half-life (≈human VISTA knock-in (24 to 27 hours or less) in rodents. In an exemplary embodiment, the subject ADCs have a rapid onset of action and are so effectively internalized by large numbers of immune cells that they are cleaved releasing large amounts of active steroid payload. , effective for a long time. The invention also relates to the use of such ADCs and novel steroids for the treatment of autoimmune, allergic and inflammatory conditions. The present invention further provides the use of such ADCs to target immune cells such as monocytes, neutrophils, T cells, Tregs, eosinophils, macrophages, dendritic cells, NK cells, particularly myeloid cells. Reduce the adverse side effects of glucocorticoids and/or improve their efficacy by selectively delivering anti-inflammatory agents to non-target cells, thereby reducing the potential otherwise induced by steroid compounds to non-target cells. The present invention relates to a method for reducing the toxicity of toxic substances.

Specifically, herein, anti-VISTA antibodies or antibody fragments typically have very short serum half-lives at physiological conditions (≈pH 7.5); 5) in human VISTA knock-in rodents for 72 hours, 1 to 32 hours, 1 to 16 hours, 1 to 8 hours, 1 to 4 hours, or 1 to 2 hours ± 0.5 hours, and in primates (cynomolgus monkeys) An antibody or antibody fragment having a serum half-life of ≈3.5, 3, 2.5, or 2.3 days ± 0.5 days and formulas (I), (II), or (III), optionally with a linker, e.g., a peptide or non-peptide linker, which may optionally be cleavable under certain conditions, e.g. a glucocorticoid receptor agonist attached via an esterase-cleavable dipeptide linker and optionally directly or indirectly attached to the antibody via a heterobifunctional or heterotrifunctional group; ADCs are provided which, when administered to a subject in need thereof, target such glucocorticoid receptor agonists to target immune cells, e.g., monocytes, T cells, neutrophils. , Tregs, CD8 T cells, CD4 T cells, eosinophils, dendritic cells, NK cells, macrophages, or myeloid cells, resulting in functional internalization of the glucocorticoid receptor agonist therein; The desired inhibitory effect on inflammation is elicited without or with substantially reduced adverse side effects such as toxicity to non-target cells. Further provided are methods of making and using such ADCs, particularly for use in treating autoimmune, allergic, and inflammatory conditions such as those previously identified.

Ｘが、フェニル、スピロ［３．３］ヘプタン、３～６員複素環、シクロアルキル、スピロアルキル、スピロヘテロシクロアルキル、二環式アルキル、ヘテロ二環式アルキル、［１．１．１］ビシクロペンタン、ビシクロ［２．２．２］オクタン、アダマンタン、及びキュバンから選択され、これらの各々を、独立して、Ｆ、Ｃｌ、Ｂｒ、Ｉ、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換することができ、その環構造の各々が、Ｎ、Ｓ、及びＯから選択される少なくとも１つの骨格ヘテロ原子を含み得、任意選択的に、１～４個のＣ１～３アルキルまたはＣ１～３ペルフルオロアルキルで更に置換され、
Ｚが、フェニル、スピロ［３．３］ヘプタン、３～６員複素環、シクロアルキル、スピロアルキル、スピロヘテロシクロアルキル、二環式アルキル、ヘテロ二環式アルキル、［１．１．１］ビシクロペンタン、ビシクロ［２．２．２］オクタン、アダマンタン、及びキュバンから選択され、これらの各々を、独立して、Ｆ、Ｃｌ、Ｂｒ、Ｉ、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換することができ、その環構造の各々が、Ｎ、Ｓ、及びＯから選択される少なくとも１つの骨格ヘテロ原子を含み得、任意選択的に、１～４個のＣ１～３アルキルまたはＣ１～３ペルフルオロアルキルで更に置換され、
Ｙが、ＣＨＲ１、Ｏ、Ｓ、及びＮＲ１から選択され、
Ｅが、ＣＨ２及びＯから選択され、
Ｇが、ＣＨ及びＮから選択され、
更に、ＧがＣＨであり、Ｘがフェニルであるときに、Ｚがフェニルではなく、
ＸへのＧの結合が、任意選択的にＣ１～３アルキル及びエチレンオキシドから選択され得、これらの各々が、独立して、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換され得、任意選択的に、１～４個のＣ１～３アルキルで更に置換され、
ＺへのＸの結合が、Ｘ及びＺ上の任意の利用可能な位置を占有し得、
置換基ＮＲ１Ｒ２が、Ｚ上の任意の利用可能な位置を占有し得、
Ｒ１が、Ｈ、１～８個の炭素の直鎖または分岐アルキル、アリール、及びヘテロアリール基から選択され、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｒ１がＨであるとき、Ｒ２が、Ｈ、１～８個の炭素の直鎖または分岐アルキル、アリール、及びヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｒ１がＨ、１～８個の炭素の直鎖もしくは分岐アルキル、またはヘテロアリールであるとき、Ｒ２が、
［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］ｍ－［Ｃ＝Ｏ］－［Ｖ］ｋ－Ｊ、
Ｃ＝Ｏ）ＯＣＨ２－ｐ－アミノフェニル－Ｎ－Ｖ－Ｊ、
（Ｃ＝Ｏ）ＯＣＨ２－ｐ－アミノフェニル－Ｎ－［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］ｍ－［Ｃ＝Ｏ］－［Ｖ］ｋ－Ｊ、及び
［Ｖ］ｋ－（Ｃ＝Ｏ）ＯＣＨ２－ｐ－アミノフェニル－Ｎ－［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］ｍ－［Ｃ＝Ｏ］－Ｊ、から選択される官能基であり得、
式中、ｍ＝１～６であり、ｋ＝０～１であり、Ｗの各順列が、独立して、Ｈ、ｎ＝１～４である［（ＣＨ２）ｎＲ３］、Ｒ３で終端する分岐アルキル鎖、及び１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基から選択され得、
Ｒ３が、Ｈ、メチル、エチル、イソプロピル、ＯＨ、Ｏ－アルキル、ＮＨ２、ＮＨ－アルキル、Ｎ－ジアルキル、ＳＨ、Ｓ－アルキル、グアニジン、尿素、カルボン酸、カルボキサミド、カルボン酸エステル、置換または非置換アリール、置換または非置換ヘテロアリールから選択され、当該アリール及びヘテロアリール置換基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択され得、
Ｖが、１～８個の炭素のアルキル鎖、１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基、１～８個の炭素を含む直鎖または分岐アルキル基、－Ｏ－アルキル、カルボン酸、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－、１～３アミノ酸配列であって、各アミノ酸が、独立して、Ｇｌｕ、Ｇｌｙ、Ａｓｎ、Ａｓｐ、Ｇｌｎ、Ｌｅｕ、Ｌｙｓ、Ａｌａ、βＡｌａ、Ｐｈｅ、Ｖａｌ、及びＣｉｔから選択される、１～３アミノ酸配列、アリール、ならびにヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｊが、－ＮＨ２、Ｎ３、チオ、シクロオクチン、－ＯＨ、－ＣＯ２Ｈ、トランスシクロオクテン、アルキニル、プロパルギル、

から選択される反応基であり、
式中、Ｒ３２が、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ３３が、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ３４が、Ｈ、Ｍｅ、テトラジン－Ｈ、及びテトラジン－Ｍｅであり、
Ｒ５が、－ＣＨ２ＯＨ、－ＣＨ２ＳＨ、－ＣＨ２Ｃｌ、－ＳＣＨ２Ｃｌ、－ＳＣＨ２Ｆ、－ＳＣＨ２ＣＦ３、ヒドロキシ、－ＯＣＨ２ＣＮ、－ＯＣＨ２Ｃｌ、－ＯＣＨ２Ｆ、－ＯＣＨ３、－ＯＣＨ２ＣＨ３、－ＳＣＨ２ＣＮ、及び

からなる群から選択され、
Ｒ６及びＲ７が、独立して、水素及びＣ１～１０アルキルから選択され、
Ｑが、Ｈ、

Ｒ８が１～８個の炭素の直鎖もしくは分岐アルキルである、Ｃ（Ｏ）Ｒ８、またはｎ＝１～４であり、Ｒ４＝Ｈ、アルキルもしくは分岐アルキル、またはＰ（Ｏ）ＯＲ４である、（Ｃ＝Ｏ）ＮＲ４ＣＨｎＮＲ４（Ｃ＝Ｏ）ＯＣＨ２－（Ｖ）ｎ－Ｊであり得、
Ａ１及びＡ２が、独立して、Ｈ及びＦから選択され、
別途明記しない限り、全ての可能な立体異性体が特許請求される、グルココルチコイドアゴニスト化合物が提供される。 More specifically, a glucocorticoid agonist compound having the following structure of formula (I),

X is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C1 to further substituted with 3 alkyl or C1-3 perfluoroalkyl,
Z is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C1 to further substituted with 3 alkyl or C1-3 perfluoroalkyl,
Y is selected from CHR1, O, S, and NR1;
E is selected from CH2 and O;
G is selected from CH and N;
Furthermore, when G is CH and X is phenyl, Z is not phenyl,
The bond of G to optionally further substituted with 1 to 4 C1-3 alkyl,
the bond of X to Z may occupy any available position on X and Z;
The substituent NR1R2 may occupy any available position on Z;
R1 is selected from H, linear or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and the aryl and heteroaryl groups are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH , -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O) -, and dialkylamino C(O)-,
When R1 is H, R2 may be selected from H, straight chain or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and the aryl and heteroaryl groups are alkyl, haloalkyl, halogen, Biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O- , alkylamino-C(O)-, and dialkylamino-C(O)-,
When R1 is H, linear or branched alkyl of 1 to 8 carbons, or heteroaryl, R2 is
[(C=O)CH(W)NH]m-[C=O]-[V]kJ,
C=O)OCH2-p-aminophenyl-NVJ,
(C=O)OCH2-p-aminophenyl-N-[(C=O)CH(W)NH]m-[C=O]-[V]k-J, and [V]k-(C= O) OCH2-p-aminophenyl-N-[(C=O)CH(W)NH]m-[C=O]-J,
where m=1 to 6, k=0 to 1, and each permutation of W is independently H, n=1 to 4 [(CH2)nR3], a branch terminating in R3 may be selected from alkyl chains, and linear or branched polyethylene oxide groups containing from 1 to 13 units;
R3 is H, methyl, ethyl, isopropyl, OH, O-alkyl, NH2, NH-alkyl, N-dialkyl, SH, S-alkyl, guanidine, urea, carboxylic acid, carboxamide, carboxylic acid ester, substituted or unsubstituted selected from aryl, substituted or unsubstituted heteroaryl, wherein the aryl and heteroaryl substituents are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkylamino, may be selected from thiols, thioalkyls, guanidines, ureas, carboxylic acids, alkoxyls, carboxamides, carboxylic esters, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)-;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein the aryl and The heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl- may be substituted with a functional group selected from C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH2, N3, thio, cyclooctyne, -OH, -CO2H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R32 is selected from Cl, Br, F, mesylate, and tosylate, and R33 is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-(4- nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, R34 is H, Me, tetrazine-H, and tetrazine-Me;
R5 is -CH2OH, -CH2SH, -CH2Cl, -SCH2Cl, -SCH2F, -SCH2CF3, hydroxy, -OCH2CN, -OCH2Cl, -OCH2F, -OCH3, -OCH2CH3, -SCH2CN, and

selected from the group consisting of
R6 and R7 are independently selected from hydrogen and C1-10 alkyl;
Q is H,

R8 is straight chain or branched alkyl of 1 to 8 carbons, C(O)R8, or n=1 to 4 and R4=H, alkyl or branched alkyl, or P(O)OR4; (C=O)NR4CHnNR4(C=O)OCH2-(V)n-J;
A1 and A2 are independently selected from H and F;
Unless otherwise specified, glucocorticoid agonist compounds are provided in which all possible stereoisomers are claimed.

式中、
Ｙが、ＣＨ２及びＯから選択され、
Ｅが、ＣＨ２及びＯから選択され、
Ｇが、ＣＨ及びＮから選択され、
Ｌが、Ｈ及びＦから選択され、
Ｒ５が、－ＣＨ２ＯＨ、－ＳＣＨ２Ｆ、及び

から選択され、
Ａ１及びＡ２が、独立して、Ｈ及びＦから選択され、
Ｖが、１～８個の炭素のアルキル鎖、１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基、１～８個の炭素を含む直鎖または分岐アルキル基、－Ｏ－アルキル、カルボン酸、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－、１～３アミノ酸配列であって、各アミノ酸が、独立して、Ｇｌｕ、Ｇｌｙ、Ａｓｎ、Ａｓｐ、Ｇｌｎ、Ｌｅｕ、Ｌｙｓ、Ａｌａ、βＡｌａ、Ｐｈｅ、Ｖａｌ、及びＣｉｔから選択される、１～３アミノ酸配列、アリール、ならびにヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｊが、－ＮＨ２、Ｎ３、チオ、シクロオクチン、－ＯＨ、－ＣＯ２Ｈ、トランスシクロオクテン、アルキニル、プロパルギル、

から選択される反応基であり、
式中、Ｒ_３２が、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ３３が、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ３４が、Ｈ、Ｍｅ、テトラジン－Ｈ、及びテトラジン－Ｍｅである、グルココルチコイドアゴニスト化合物が提供される。 Furthermore, a glucocorticoid agonist compound having the structure of formula (II),

During the ceremony,
Y is selected from CH2 and O;
E is selected from CH2 and O;
G is selected from CH and N;
L is selected from H and F;
R5 is -CH2OH, -SCH2F, and

selected from
A1 and A2 are independently selected from H and F;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein the aryl and The heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl- may be substituted with a functional group selected from C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH2, N3, thio, cyclooctyne, -OH, -CO2H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R ₃₂ is selected from Cl, Br, F, mesylate, and tosylate, and R 33 is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-(4 -nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R34 is H, Me, tetrazine-H, and tetrazine-Me.

式中、
Ｙが、ＣＨ２及びＯから選択され、
Ｅが、ＣＨ２及びＯから選択され、
Ｇが、ＣＨ及びＮから選択され、
Ｌが、Ｈ及びＦから選択され、
Ｒ５が、－ＣＨ２ＯＨ、－ＳＣＨ２Ｆ、及び

から選択され、
Ａ１及びＡ２が、独立して、Ｈ及びＦから選択され、
Ｖが、１～８個の炭素のアルキル鎖、１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基、１～８個の炭素を含む直鎖または分岐アルキル基、－Ｏ－アルキル、カルボン酸、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－、１～３アミノ酸配列であって、各アミノ酸が、独立して、Ｇｌｕ、Ｇｌｙ、Ａｓｎ、Ａｓｐ、Ｇｌｎ、Ｌｅｕ、Ｌｙｓ、Ａｌａ、βＡｌａ、Ｐｈｅ、Ｖａｌ、及びＣｉｔから選択される、１～３アミノ酸配列、アリール、ならびにヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｊが、－ＮＨ２、Ｎ３、チオ、シクロオクチン、－ＯＨ、－ＣＯ２Ｈ、トランスシクロオクテン、アルキニル、プロパルギル、

から選択される反応基であり、
式中、Ｒ３２が、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ３３が、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ３４が、Ｈ、Ｍｅ、テトラジン－Ｈ、及びテトラジン－Ｍｅである、グルココルチコイドアゴニスト化合物も提供される。 Further, a glucocorticoid agonist compound having the structure of formula (III),

During the ceremony,
Y is selected from CH2 and O;
E is selected from CH2 and O;
G is selected from CH and N;
L is selected from H and F;
R5 is -CH2OH, -SCH2F, and

selected from
A1 and A2 are independently selected from H and F;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein the aryl and The heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH2, alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl- may be substituted with a functional group selected from C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH2, N3, thio, cyclooctyne, -OH, -CO2H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R32 is selected from Cl, Br, F, mesylate, and tosylate, and R33 is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-(4- Also provided are glucocorticoid agonist compounds selected from (nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R34 is H, Me, tetrazine-H, and tetrazine-Me.

Also provided are glucocorticoid agonist linker compounds comprising a glucocorticoid agonist having the structure of Formula (I), (II), or (III) attached to a cleavable or non-cleavable linker.

Furthermore, an immune cell antigen, such as VISTA, attached to a glucocorticoid agonist having the structure of formula (I), (II), or (III), which in turn is attached to a cleavable or non-cleavable linker. An ADC is provided that includes an antibody that binds to.

Additionally, compositions and medicaments are provided that include the glucocorticoid agonists, glucocorticoid agonist linkers, and ADCs containing them.

Further provided are therapeutic and prophylactic uses of such glucocorticoid agonists, glucocorticoid agonist linkers, and ADCs containing them, particularly for treating inflammatory, allergic, and autoimmune conditions.

Unless otherwise defined, as commonly understood, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the present invention or in testing the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. The nomenclature utilized in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, as well as their experimental procedures and techniques, described herein are well known and commonly used in the art. It is something that exists. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

Definitions To facilitate understanding of this disclosure, several terms and phrases are defined below.

As used throughout this description and the following claims, the meanings of "a," "an," and "the" refer to multiple references unless the context clearly indicates otherwise. include.

In this disclosure, the term "glucocorticosteroid" or "steroid" refers to naturally occurring or synthetic steroid hormones that interact with glucocorticoid receptors. Non-limiting exemplary glucocorticoid steroids include, among others, those described in WO2009/069032, US20180126000, WO05/028495, preferably novel glucocorticoid agonists of formula I, II, or III. , as well as glucocorticoid agonist linkers and ADCs containing the same glucocorticoid agonists disclosed herein. Non-limiting examples of known glucocorticosteroids include:

Other known glucocorticosteroids are described in WO2009/069032. Specific examples of glucocorticosteroids include 16-alpha hydroxyprednisolone, dexamethasone, difluorasone, flumethasone, flunisolide, fluocinolone acetonide, fluticasone propionate, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, triamcinolone acetonide, and Included are novel glucocorticoid agonists of formula I, II or III, as well as glucocorticoid agonist linkers and ADCs comprising the same glucocorticoid agonists disclosed herein.

A "glucocorticosteroid derivative" refers to the addition of one or more atoms or functional groups to facilitate attachment of the "glucocorticosteroid derivative" to another moiety, such as a linker and/or an antibody or antibody fragment. or a compound derived by removal. In general, this addition or removal does not interfere with the activity of the "glucocorticosteroid derivative", ie, its ability to induce anti-inflammatory activity upon internalization by immune cells. "Glucocorticosteroid derivative" specifically includes "glucocorticosteroid radical" or "glucocorticosteroid radical."

“Glucocorticosteroid radical” or “glucocorticosteroid radical” is a term used to describe the term “glucocorticosteroid radical” or “glucocorticosteroid radical” used to describe Produced by the removal of one or more atoms, namely hydrogen atoms. For example, a hydrogen atom may be removed from any suitable _-NH2 group of the parent glucocorticosteroid, and a hydrogen atom may be removed from any suitable -OH group of the parent glucocorticosteroid. , the hydrogen atom may be removed from any suitable -SH group, the hydrogen atom may be removed from any suitable -N(H)- group, and the hydrogen atom may be removed from the parent glucorticosteroid from any suitable -CH ₃ , -CH ₂ , or -CH= group.

In this disclosure, the term "heterobifunctional" or "heterotrifunctional" refers to a linker and a chemical moiety (disclosed herein) that can optionally be used to connect an anti-VISTA antibody or antibody fragment. In the general formula of ADC, it refers to (“Q”)). Heterobi- and trifunctional groups are characterized by having different reactive groups at each end of the chemical moiety. Non-limiting exemplary heterobifunctional groups are disclosed in U.S. Publication No. 20180126000, which is incorporated herein by reference, and in the Exemplary Embodiments section and Examples, e.g., Example 3 of this application. Further exemplified by the disclosed ADC conjugates and the compounds of Figures 118A-O of this application.

例示的なヘテロ三官能基

Heterobi- and trifunctional groups are well known in the art for specifically producing protein conjugates and antibody drug conjugates (ADCs). These moieties are characterized by having different reactive groups at both ends of the chemical moiety. Non-limiting exemplary heterobifunctional groups include:

Exemplary heterotrifunctional groups

As used herein, the terms "antibody" and "antibodies" are technical terms and can be used interchangeably herein and refer to an antigen-binding site that specifically binds to an antigen. Refers to molecules that have

The term "antibody" refers to the recognition and specificity of a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one antigen recognition site within the variable region of an immunoglobulin molecule. means an immunoglobulin molecule that binds to As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion antibodies, so long as the antibody exhibits the desired biological activity. proteins, and any other modified immunoglobulin molecules. Antibodies belong to any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and It can be IgM, or a subclass (isotype) thereof (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different classes of immunoglobulins have different well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc. As used herein, the term "antibody" includes bispecific and multispecific antibodies.

The term "antibody fragment" refers to a portion of an intact antibody. "Antigen-binding fragment" refers to the portion of an intact antibody that binds an antigen. Antigen-binding fragments can include antigen-determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , and Fv fragments, linear antibodies, and single chain antibodies. An "antigen-binding fragment" can be a bispecific or multispecific antigen-binding fragment.

A "blocking" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds, such as VISTA. In some embodiments, blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. Biological activity can be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

A "promoting" or "enhancing" antibody, an "agonist" antibody is an antibody that enhances or increases the biological activity of the antigen to which it binds, such as VISTA. In some embodiments, blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. Biological activity can be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

The term "anti-VISTA antibody" or "antibody that binds to VISTA" refers to an antibody that binds to VISTA, generally human VISTA, with sufficient affinity so that the antibody is useful for targeting VISTA-expressing immune cells. Refers to an antibody that specifically binds to. The extent of binding of an anti-VISTA antibody to an unrelated non-VISTA protein can be less than about 10% of the binding of the antibody to VISTA, for example, as measured by radioimmunoassay (RIA). In certain embodiments, the antibody that binds VISTA has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. Exemplary anti-VISTA antibodies and fragments included in the subject ADCs contain the same CDRs and/or the same variable heavy and light chain polypeptides as those of VSTB94 or VSTB49-116, i.e., FIGS. 8, 10 and 10, respectively. It would have the arrangement shown in FIG.

A "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous population of antibodies or antigen-binding fragments that engage in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof refers to both intact and full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab') ₂ , Fv, etc.), single chain (scFv) variants. , fusion proteins containing antibody portions, and any other modified immunoglobulin molecules containing antigen recognition sites. Additionally, "monoclonal" antibodies or antigen-binding fragments thereof include such antibodies and their antigens produced in any number of ways, including, but not limited to, hybridomas, phage selection, recombinant expression, and transgenic animals. Refers to combined fragments.

The term "humanized" antibody or antigen-binding fragment thereof is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof that contains minimal non-human (e.g., murine) sequence. Refers to the form of an antibody or antigen-binding fragment. Typically, humanized antibodies or antigen-binding fragments thereof are derived from a non-human species (e.g., mouse, rat, A human immunoglobulin that is replaced (“CDR grafted”) by residues from the CDRs of a human immunoglobulin (Rabbit, Hamster) (Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332 :323-327 (1988), Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, Fv framework region (FR) residues of a human immunoglobulin are replaced with corresponding residues in an antibody or fragment from a non-human species that has the desired specificity, affinity, and potency. . The humanized antibody or antigen-binding fragment thereof is further modified by substitution of additional residues, either within the Fv framework regions and/or within the replaced non-human residues, to improve the specificity of the antibody or antigen-binding fragment thereof. , affinities, and/or capabilities can be refined and optimized. Generally, a humanized antibody or antigen-binding fragment thereof comprises at least one, and typically two or three, substantially all of the CDR regions corresponding to a non-human immunoglobulin. while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. The humanized antibody or antigen-binding fragment thereof can also include at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in US Pat. No. 5,225,539, Roguska et al. , Proc. Natl. Acad. Sci. , USA, 91(3):969-973 (1994), and Roguska et al. , Protein Eng. 9(10):895-904 (1996). In some embodiments, a "humanized antibody" is a resurfaced antibody.

A "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are brought together in close proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes) of Health, Bethesda Md.)), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec. Biol. 273:927-948). Additionally, a combination of these two approaches is sometimes used in the art to determine CDRs.

The Kabat numbering system is commonly used to refer to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain). (For example, Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Unless explicitly stated otherwise, the numbering system used herein is the Kabat numbering system.

Kabat-like amino acid position numbering is based on Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) refers to the numbering system used for the heavy or light chain variable domains of a collection of antibodies. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions into the FRs or CDRs of the variable domain. For example, the heavy chain variable domain contains a single amino acid insertion after residue 52 of H2 (residue 52a by Kabat), and an inserted residue after heavy chain FR residue 82 (e.g., by Kabat). residues 82a, 82b, and 82c). Kabat numbering of residues can be determined for a given antibody by alignment in regions of homology of the antibody's sequences with "standard" Kabat numbered sequences. Chothia instead refers to the position of a structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme and H35B, and if neither 35A or 35B is present, the loop terminates at 32; if only 35A is present, the loop terminates at 33; and if both 35A and 35B are present, The loop ends at 34). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the Chothia numbering scheme, which refers to the position of immunoglobulin structural loops (e.g., Chothia C & Lesk A M, (1987), J Mol Biol 196 :901-917, Al-Lazikani B et al., (1997) J Mol Biol 273:927-948, Chothia C et al., (1992) J Mol Biol 227:799-817, Tramontano A et al. al., ( 1990) J Mol Biol 215(1):175-82 and US Pat. No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26-32, 33, or 34, and the Chothia CDR-H2 loop is present at heavy chain amino acids 52-56. The Chothia CDR-H3 loop is present at heavy chain amino acids 95-102, while the Chothia CDR-L1 loop is present at light chain amino acids 24-34, and the Chothia CDR-L2 loop is present at light chain amino acids 50. ~56 and the Chothia CDR-L3 loop resides at light chain amino acids 89-97. The ends of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme and H35B, if neither 35A or 35B is present, the loop terminates at 32; if only 35A is present, the loop terminates at 33; and if both 35A and 35B are present, The loop ends at 34).

In certain embodiments, the CDRs of antibodies or antigen-binding fragments thereof are as described in Lefranc MP, (1999) The Immunologist 7:132-136, and Lefranc MP et al. , (1999) Nucleic Acids Res 27:209-212. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26-35, VH-CDR2 is located at positions 51-57, VH-CDR3 is located at positions 93-102, and VL-CDR1 is located at positions 27-102. VL-CDR2 is in the 50th to 52nd position, and VL-CDR3 is in the 89th to 97th position.

In certain embodiments, the CDRs of the antibody or antigen-binding fragment thereof are those described by MacCallum R M et al. , (1996) J Mol Biol 262:732-745. Also, for example, Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Konterman and Dubel, eds. , Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

In certain embodiments, the CDRs of the antibody or antigen-binding fragment thereof represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). It can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions.

A "constant region" of an antibody refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, either alone or in combination.

The term "human" antibody means an antibody produced by a human, or an antibody made using any technique known in the art, that has an amino acid sequence corresponding to an antibody produced by a human. do. This definition of human antibodies includes intact or full-length antibodies, fragments thereof, and/or antibodies that contain at least one human heavy and/or light chain polypeptide, such as antibodies that contain a murine light chain and a human heavy chain polypeptide. etc. are included.

The term "chimeric" antibody refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from more than one species. Typically, both the light and heavy chain variable regions of an antibody are derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and potency. While corresponding to the variable regions, the constant regions are homologous to sequences in antibodies derived from another species (usually humans) to avoid eliciting an immune response in that species.

The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen that is capable of being recognized by and specifically binding to a particular antibody. When the antigen is a polypeptide, epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, while epitopes formed by tertiary folding are typically lost upon protein denaturation. Epitopes typically contain at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial configuration. Preferred epitopes on VISTA to which exemplary anti-VISTA antibodies can bind are identified in FIG. 10.

"Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). . The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and remain bound longer. Tend. Various methods of measuring binding affinity are known in the art, any of which can be used for purposes of this disclosure. Such methods include surface plasmon resonance (BIAcore), ELISA, Kinexa Biosensor, scintillation proximity assay, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display. Binding affinity can also be screened using a suitable bioassay. In this application, the Kd of exemplary anti-VISTA antibodies included in exemplary ADCs was determined by surface plasmon resonance (SPR) method on a ProteOn instrument.

"Or better," as used herein to refer to binding affinity, refers to stronger binding between a molecule and its binding partner. "Greater than" as used herein refers to stronger binding, represented by a smaller number of Kd values. For example, an antibody having an affinity for an antigen of "greater than or equal to 0.6 nM," where the antibody's affinity for the antigen is <0.6 nM, i.e., 0.59 nM, 0.58 nM, 0.57 nM, etc., or 0. Any value less than .6 nM.

"Specifically binds" generally means that an antibody binds to an epitope through its antigen-binding domain, and that binding involves some degree of complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope more readily through its antigen-binding domain than it binds to a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity with which a particular antibody binds a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" may be considered to have higher specificity for a related epitope "D." can also be said to bind to epitope "C" with high specificity.

"Preferentially binds" means that the antibody specifically binds to an epitope more readily than it binds to a related, similar, homologous, or similar epitope. Thus, an antibody that "preferentially binds" to a given epitope will be more likely to bind to that epitope than to a related epitope, even though such antibodies may cross-react with related epitopes.

An antibody "competitively inhibits" the binding of a reference antibody to a given epitope if the antibody preferentially binds to that epitope or an overlapping epitope to the extent that it blocks the binding of the reference antibody to the epitope. ” is said. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. An antibody can be said to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

"Isotype" herein refers to the antibody class (eg, IgM, IgG1, IgG3, IgG3, or IgG4) encoded by the heavy chain constant region genes.

While "K-assoc" or "Ka" as used herein broadly refers to the association rate of a particular antibody-antigen interaction, "Kdiss" or "Kd '' refers to the dissociation rate of a particular antibody-antigen interaction.

The term "KD" as used herein refers to the dissociation constant, obtained from the ratio of Kd to Ka (ie, Kd/Ka) and expressed as molar concentration (M). KD values for antibodies can be determined using well-established methods in the art such as plasmon resonance (BIAcore®), ELISA and KINEXA. Preferred methods for determining the KD of an antibody are using surface plasmon resonance, preferably by using a biosensor system such as the BIAcore® system, or by ELISA. Typically these methods are accomplished at 25°C or 37°C. Antibodies for therapeutic use will generally possess a KD as determined by surface plasmon resonance of 50 nM or less, or more typically 1 nM or less, at 25°C or 37°C.

The phrase "Kd" herein refers to the equilibrium dissociation constant, which is the calculated ratio of Koff/Kon between an antibody and its antigen. Association constant (Kon) is used to characterize how quickly an antibody binds to its target. Herein, antibody Kd was determined by surface plasmon resonance (SPR) using a Proteon instrument.

The term "PK" as used herein refers to an antibody or antibody fragment or antibody drug conjugate (ADC), preferably an anti-VISTA or antibody fragment according to the invention (i.e., an anti-VISTA antibody that binds to VISTA-expressing cells at physiological pH). VISTA antibodies or antibody fragments) and anti-inflammatory agents (AIs), small molecules that require cellular internalization for efficacy (anti-inflammatory activity), typically steroids, more typically refers to the in vivo half-life or duration (in hours) that half of the amount of AI, a glucorticosteroid agonist of formula I, II or III, remains in the peripheral circulation in the serum. PK can be determined in vivo in subjects administered the antibody or antibody fragment or ADC, e.g., human VISTA knock-in rodents, or in primates (e.g., humans or cynomolgus monkeys), as described below. Anti-VISTA antibodies included in ADCs according to the invention typically have a short PK, i.e., typically about 2.3 ± 0.7 days in cynomolgus monkeys, and typically about 2.3 ± 0.7 days in human VISTA knock-in rodents. , will include at most ≈2.5 days, more typically only one day, no more than a few hours.

The term "PD" as used herein refers to antibodies or antibody drug conjugates (ADCs) according to the invention, such as those comprising anti-VISTA antibodies or antibody fragments that bind to VISTA-expressing cells at physiological pH, and anti-inflammatory agent (AI) that requires cellular internalization for efficacy (anti-inflammatory activity), typically a steroid, more typically of formula I, II or III. Refers to the duration (hours) during which a dose of an AI, including a glucorticosteroid agonist, induces efficacy (anti-inflammatory activity) upon internalization into target cells. The PD of a steroid, eg, a glucocorticosteroid agonist of Formula I, II or III, or a glucocorticoid agonist linker disclosed herein and an ADC containing the same linker, can be determined by different assays. For example, the PD of a VISTA ADC according to the invention can be determined in vitro using VISTA-expressing immune cells contacted with the ADC or in a subject administered an ADC dose, e.g., in a rodent or primate ( for example, in humans or cynomolgus monkeys). In addition, because the exemplary anti-VISTA ADCs bind to different immune cells (e.g., T cells, Tregs, monocytes, macrophages, neutrophils), these ADCs also bind to different types of immune cells. Based on the relative expression of the antigen bound thereby, e.g. VISTA expression, different types of immune cells internalize the immune cells in question differently and, furthermore, because the turnover rate of such immune cells changes. PD values when determined in vitro using immune cells, eg, VISTA expressing immune cells, will vary. Generally, herein, in the case of anti-VISTA ADCs, PD is expressed based on the duration of anti-inflammatory activity induced by macrophages, since these cells are present in the circulation, and (surprisingly) ) VISTA antibodies comprising ADCs according to the invention have been demonstrated to induce long-term anti-inflammatory activity in macrophages, for example for several weeks or even a month after ADC administration. However, if the ADC targets different immune cells than VISTA, e.g. B cells or NK cells, it is difficult to detect inflammatory activity within these cells since the internalization of glucocorticoid agonists is within these cells. would potentially determine the PD.

The phrase "PD/PK ratio" as used herein refers, for example, to human VISTA knock-in anti-VISTA ADCs in rodents or primates (e.g., humans or cynomolgus monkeys), in immune cells of a particular species, or in animal In a model, it refers to the ratio of PD and PK values of an ADC according to the invention, determined in vitro or in vivo. [As shown below, the PD/PK ratio of anti-VISTA ADCs according to the invention is surprisingly high, i.e. values as high as 14:1 or 28:1 have been demonstrated in VISTA knock-in rodents and cynomolgus monkeys. There is. Furthermore, because the expression of VISTA by different immune cells in rodents and humans and primates is very similar, drug metabolism is generally much more complex in rodents than in humans and non-human primates. occurs rapidly, and similar or higher PD/PK ratios are expected to be obtained in humans and other non-human primates. Although Applicants do not wish to be bound by this theory, in the case of VISTA antibodies comprising ADCs according to the invention, the ADCs of interest are present in very large quantities due to the high density of surface VISTA expression on these immune cells. Internalizing a specific type of VISTA-expressing cell apparently produces a "depot effect", i.e. the depot of internalized ADC is very slowly metabolized, thereby reducing therapeutically effective (anti-inflammatory) amounts. It is believed to provide surprisingly long-term release of anti-inflammatory agents (eg, steroids such as glucorticosteroid agonists of Formula I, II, or III, or glucocorticosteroid agonist linkers, or ADCs containing the same).

"Onset of efficacy" refers to the time in vivo when the efficacy of a therapeutic agent, eg, steroid or ADC conjugate, begins. In the present invention, this is done using known in vivo assays that detect the anti-inflammatory effects of steroids, such as glucocorticosteroid agonists of formula I, II or III, or glucocorticosteroid agonist linkers, or ADCs according to the invention. can be detected in a subject who has been administered the conjugate. As disclosed below, anti-VISTA ADCs according to the invention have been shown to have a rapid onset of efficacy, ie, about 2 hours, in human VISTA knock-in rodents.

As used herein, the phrases "substantially similar" or "substantially the same" can be understood by those skilled in the art to refer to two values (generally one value and one value associated with the antibodies of the present disclosure). the other value relative to the reference/comparison antibody) has little biological and/or statistical significance within the context of the biological property measured by that value (e.g., Kd value). represents a sufficiently high degree of similarity between two numbers such that they are considered to be equal or not present at all. The difference between the two values can be less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value of the reference/comparison antibody.

An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form that is not found in nature. An isolated polypeptide, antibody, polynucleotide, vector, cell or composition includes one that has been purified to the extent that it is no longer in the form in which it is found in nature. In some embodiments, the isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

As used herein, "substantially pure" means at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least Refers to a substance that is 99% pure.

The term "immunoconjugate,""conjugate,""antibody drug conjugate," or "ADC" as used herein refers to a compound or derivative thereof that is attached to an anti-VISTA antibody or fragment thereof. ), and anti-inflammatory agents such as glucocorticosteroid agonists, and generally refers to the intervening linker, which may be represented by the following general formula: (AI-L-Q) _n- A, where AI= anti-inflammatory agents, generally small molecule glucocorticoid receptor agonists, such as glucocorticosteroid agonist compounds according to formula 1, II or III disclosed herein, L = linker, Q = heterobifunctional group, a heterotrifunctional group, or absent; A = an anti-VISTA antibody or VISTA-binding fragment thereof that binds preferentially to human VISTA at physiological pH and generally possesses a short pK as described above; n is an integer greater than 1, optionally from 1 to 10. Immunoconjugates can also be defined in reverse order by the general formula below. A-(Q-L-AI) _n .

In this disclosure, the term "linker" refers to linking an antibody or antibody fragment (e.g., antigen-binding fragment) or functional equivalent thereof to an anti-inflammatory agent, generally a glucocorticosteroid receptor agonist, e.g., Formula I , II, or III glucocorticosteroid agonist. The linker may be susceptible to cleavage (a "cleavable linker"), thereby facilitating the release of anti-inflammatory agents such as glucocorticosteroids. For example, such cleavable linkers can be used to target neutrophils, monocytes, macrophages, eosinophils, T cells, dendritic cells, Tregs, NK cells, B cells, mast cells, among other immune cell types. acid-induced cleavage, light-induced cleavage, peptidase induction under conditions in which glucocorticosteroids and/or antibodies remain active before or after internalization into immune cells such as , macrophages, or bone marrow cells. may be susceptible to sexual cleavage, esterase-induced cleavage, and disulfide bond cleavage. Alternatively, the linker may be substantially resistant to cleavage (a "non-cleavable linker").

As a non-cleavable linker, it is possible to attach an anti-inflammatory agent, such as a glucocorticosteroid agonist of formula I, II or III, to the antibody in a stable covalent manner, and as described above for cleavable linkers. Any chemical moiety that does not fall into the listed categories is included. Thus, non-cleavable linkers are substantially resistant to acid-induced, light-induced, peptidase-induced, esterase-induced, and disulfide bond cleavage. Additionally, non-cleavable refers to acid-, photo-labile cleavage agents in conditions where the glucocorticosteroid and/or antibody does not lose its activity before or after internalization into immune cells such as monocytes or myeloid cells. , refers to the ability of a linker or a chemical bond adjacent to a linker to withstand cleavage induced by peptidases, elastases, or chemical or physiological compounds that cleave disulfide bonds.

Some cleavable linkers are cleaved by peptidases ("peptidase cleavable linkers"). Only certain peptides are easily cleaved inside or outside the cell. For example, Trout et al. , 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J. See Cancer, 677-684 (1989). Furthermore, peptides are composed of α-amino acid units and peptide bonds, which are chemically amide bonds between the carboxylate of one amino acid and the amino group of a second amino acid. Other amide bonds, such as the bond between a carboxylate and the alpha amino acid group of lysine, are understood not to be peptide bonds and are considered non-cleavable.

Some linkers are cleaved by esterases ("esterase cleavable linkers"). Only certain esters can be cleaved by esterases present inside or outside the cell. Esters are formed by the condensation of carboxylic acids and alcohols. Simple esters are esters produced with simple alcohols such as aliphatic alcohols and small cyclic and small aromatic alcohols.

In some embodiments, the cleavable linker moiety can include a peptide containing 1-10 amino acid residues. In these embodiments, the peptide allows cleavage of the linker by proteases, thereby promoting the release of anti-inflammatory agents, e.g., glucocorticosteroids, upon exposure to intracellular proteases such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Exemplary peptides include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include alanine-alanine (ala-ala), valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), phenylalanine - homolysine (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly), as well as those identified in the "Exemplary Embodiments" section of this application. Examples include, but are not limited to, specific linkers embodied in Example 3.

Peptides may contain naturally occurring and/or non-naturally occurring amino acid residues. The term "naturally occurring amino acids" includes Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Points to Tyr. “Unnatural amino acids” (i.e., amino acids that do not occur naturally) include, by way of non-limiting example, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, norleucine (“Nle”), These include norvaline ("Nva"), beta-alanine, L- or D-naphthalanine, ornithine ("Orn"), and the like. Peptides can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C and D, or plasmin proteases.

Amino acids also include the D form of natural and unnatural amino acids. "D-" designates an amino acid that has a "D" (dextrorotatory) configuration, as opposed to the configuration in naturally occurring ("L-") amino acids. Natural and unnatural amino acids can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

The term "drug-antibody ratio" or "DAR" refers to an anti-inflammatory agent or functional derivative (i.e., a radical derived from a small molecule glucocorticoid receptor agonist, e.g., a glucocorticosteroid of formula I, II or III). Refers to a number. Thus, in an immunoconjugate having the general formula (AI-LQ) _n -A or vice versa, the DAR is defined by the variable "n". Typically, in the subject ADC, "n" ranges from 1 to 12.

When referring to a compound having the formula (AI-L-Q) _n -A representing an individual immune complex, DAR refers to an inflammatory agent or functional derivative bound to a particular A (e.g., bound to A, A small molecule glucocorticoid receptor agonist, e.g., a radical derived from a glucocorticosteroid such as dexamethasone or budesonide, or a novel glucocorticosteroid of formula I, II, or III, e.g., where n is optionally 1 n is an integer or fraction from 1 to 12 (eg, n is an integer from 1 to 12).

When referring to a compound with the formula (AI-L-Q) _n -A representing multiple immune complexes, DAR refers to an anti-inflammatory agent or a functional derivative (e.g., a small molecule glucocorticoid attached to A by a linker). The average number of radicals (e.g., n is optionally an integer or fraction from 1 to 12) derived from a receptor agonist, e.g., a glucocorticosteroid, such as a novel steroid of Formula I, II, or III. Thus, as an example, the formula (AI-L-Q) n − includes a first immune complex with 3 AIs per A and a second immune complex with 4 _AIs per A. A compound with A would have a DAR (ie, "n") of 3.5.

The term "subject" refers to any animal (eg, mammal) that is a recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, and the like. Typically, the terms "subject" and "patient" are used interchangeably herein with reference to human subjects.

The term "pharmaceutical preparation" refers to a preparation that is in such a form as to enable the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the preparation is administered. refers to The formulation may be sterile.

An "effective amount" of an ADC or glucocorticoid receptor agonist disclosed herein is an amount sufficient to carry out the specifically stated purpose. An "effective amount" can be determined with respect to the stated purpose.

The term "therapeutically effective amount" refers to an amount of an immune complex or glucocorticoid receptor agonist effective to "treat" a disease or disorder in a subject or mammal. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result.

Terms such as "treating" or "treatment" or "cure" or "alleviation" or "alleviation" refer to curing, slowing down, or alleviating the symptoms of a diagnosed pathological condition or disorder. refers to therapeutic measures that halt the progression of and/or halt the progression of a condition or disorder. Accordingly, those in need of treatment include those who have been diagnosed with a disorder or are suspected of having a diagnosis. Preventive or precautionary measures refer to measures that prevent and/or slow down the development of a target pathological condition or disorder. Those in need of preventive or precautionary measures therefore include those who are prone to having a disability and those whose disability is to be prevented.

"Polynucleotide" or "nucleic acid" when used interchangeably herein refers to a polymer of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase. Polynucleotides can include modified nucleotides such as methylated nucleotides and analogs thereof. If present, modifications to the nucleotide structure may be applied before or after assembly of the polymer. A sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeling moieties. Other types of modifications include, for example, "caps," substitution of one or more of the naturally occurring nucleotides with analogs, internucleotide modifications, such as uncharged bonds (e.g., methylphosphonates, phosphotriesters, , phosphoramidates, carbamates, etc.) and those containing charged bonds (e.g., phosphorothioates, phosphodithioates, etc.), e.g., proteins (e.g., nucleic acids, enzymes, toxins, antibodies, signal peptides, ply-L-lysine). etc.), intercalating agents (e.g., acridine, psoralen, etc.), chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), and alkylating agents. including those containing modified linkages (eg, alpha anomeric nucleic acids, etc.), as well as unmodified form(s) of the polynucleotide. Furthermore, any of the hydroxyl groups normally present on sugars can be replaced, for example by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional bonds to additional nucleotides. or conjugated to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with an amine or organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides may also include, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, arabinose, xylose. or ribose or deoxyribose sugars as commonly known in the art, including epimeric sugars such as lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and non-basic nucleoside analogs such as methylriboside. It may also include similar forms of . One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include phosphate, P(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR ₂ (“amidate”), P(O )R, P(O)OR', CO or _CH2 ('formacetal'), each R or R' is independently H or optionally an ether (--O --) bonds, substituted or unsubstituted alkyl (1-20C), including, but not limited to, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

The term "vector" means a construct capable of delivering, and optionally expressing, one or more gene(s) or sequence(s) of interest within a host cell. . Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and producers. Examples include, but are not limited to, certain eukaryotic cells such as cells.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also refer to amino acid polymers that are naturally or interventionally modified, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation such as conjugation with a labeling moiety. Or it also includes modifications. The definition also includes, for example, polypeptides that include one or more analogs of amino acids (eg, including unnatural amino acids, etc.), as well as other modifications known in the art. Because the polypeptides of the present disclosure are antibody-based, it is understood that in certain embodiments the polypeptides can occur as a single chain or related chains.

The term "identical" or "percent identity" in the context of two or more nucleic acids or polypeptides refers to the term "identical" or "percent identity" for maximum correspondence, without considering any conservative amino acid substitutions as part of sequence identity. Refers to two or more sequences or subsequences that are the same or have a specified proportion of nucleotide or amino acid residues that are the same when compared and aligned (introducing gaps where necessary) . Percent identity can be determined using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. One such non-limiting example of a sequence alignment algorithm is Karlin et al. , Proc. Natl. Acad. Sci. , 90:5873-5877 (1993), modified in Karlin et al, Proc. Natl. Acad. Sci. , 87:2264-2268 (1990) and is incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments, gapped BLAST is performed as described by Altschul et al. , Nucleic Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, C alif.), or Megalign (DNASTAR) is an additional publicly available software program that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program of GCG software (e.g., NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 90; and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)), is used to perform the using either a Blossum 62 matrix or a PAM250 matrix and a gap weighting of 16, 14, 12, 10, 8, 6, or 4, and a length weighting of 1, 2, 3, 4, 5). Percent identity between amino acid sequences can be determined. Alternatively, in certain embodiments, percent identity between nucleotide or amino acid sequences is determined using the Myers and Miller algorithm (CABIOS, 4:11-17 (1989)). For example, percent identity can be determined using the ALIGN program (version 2.0) and using PAM120 with a residue table, a gap length penalty of 12, and a gap penalty of 4. Appropriate parameters for maximum alignment with a particular alignment software can be determined by one skilled in the art. In certain embodiments, alignment software default parameters are used. In certain embodiments, the first percent identity "X" of the first amino acid sequence to the sequence amino acids of the second is calculated as 100-fold (Y/Z), where Y is determined by visual inspection or a particular sequence alignment program. Z is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (aligned by ), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, then the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence. .

By way of non-limiting example, any particular polynucleotide has a certain percent sequence identity to a reference sequence (e.g., at least 80% identical, at least 85% identical, at least 90% identical, some implementations In certain embodiments, the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group , University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482 489 (1981)) to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, the parameters are: homology gaps of up to 5% of the total number of nucleotides in the reference sequence are set to be allowed.

In some embodiments, two nucleic acids or polypeptides of the present disclosure are substantially identical, which when compared and aligned for maximum correspondence, using a sequence comparison algorithm, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, in some embodiments at least 95%, 96%, 97%, 98%, 99%, as measured by visual inspection. nucleotide or amino acid residue identity. Identity can exist over a region of the sequence that is at least about 10, about 20, about 40-60 residues in length, or any integral value therebetween, and is at least about 60-80 residues in length. residues, e.g., at least about 90-100 residues; in some embodiments, the sequences span the entire length of the sequences being compared, e.g., coding regions of nucleotide sequences. are substantially the same.

A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non- Polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, A family of amino acid residues with similar side chains has been defined in the art, including tryptophan, histidine). For example, substitution of phenylalanine for tyrosine is a conservative substitution. In some embodiments, conservative substitutions in the sequences of polypeptides and antibodies of the present disclosure do not abolish binding of an antibody comprising an amino acid sequence to the antigen(s), eg, VISTA, to which the antibody binds. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (e.g., Brummell et al., Biochem. 32:1180-1 187 (1993), Kobayashi et al., Protein Eng. 12(10):879-884 (1999), and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

As used herein, "substantially pure" means at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, More preferably it refers to a material that is at least 98% pure, more preferably at least 99% pure.

A "host cell" includes an individual cell or cell culture that can be or has been a recipient of vector(s) for integration of a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny do not necessarily differ completely (in morphology or genomic DNA complement) from the original parent cell, whether due to natural, accidental, or intentional mutations. They may not be the same. Host cells include cells that have been transfected in vivo with polynucleotide(s) of the invention.

The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226, or from Pro230 to its carboxyl terminus. The numbering of residues within the Fc region is that of the EU index, similar to Kabat. Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. , 1991. The Fc region of an immunoglobulin generally includes two constant domains, CH2 and CH3.

As used herein, "Fc receptor" and "FcR" refer to receptors that bind to the Fc region of antibodies. A preferred FcR is a native sequence human FcR. Additionally, preferred FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcR is described by Ravetch and Kinet, 1991, Ann. Rev. Immunol. , 9:457-92, Capel et al. , 1994, ImmunoMethods, 4:25-34, and de Haas et al. , 1995, J. Lab. Clin. Med. , 126:330-41. "FcR" also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587, and Kim et al., 1994, J .Immunol., 24:249).

"Complement-dependent cytotoxicity" and "CDC" refer to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (eg, an antibody) complexed with a cognate antigen. To assess complement activation, for example, Gazzano-Santoro et al. , J. Immunol. The CDC assay can be performed as described in Methods, 202:163 (1996).

A "functional Fc region" retains at least one effector function of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, cell surface receptors (e.g. including, but not limited to, down-regulation of the B cell receptor (BCR). Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain), and various techniques are known in the art for assessing such antibody effector functions. can be evaluated using various assays.

A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" includes an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution within the native sequence Fc region or within the Fc region of the parent polypeptide as compared to the native sequence Fc region or the Fc region of the parent polypeptide, e.g. having from about 10 amino acid substitutions, preferably from about 1 to about 5 amino acid substitutions. The variant Fc regions herein preferably have at least about 80% sequence identity with the native sequence Fc region and/or the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity therewith; More preferably, it will possess at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity therewith.

As used herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to non-specific cytotoxic cells that express Fc receptors (FcR), such as natural killer (NK) cells. neutrophils, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. In vitro ADCC assays, such as those described in US Pat. No. 5,500,362 or US Pat. No. 5,821,337, can be used to assess the ADCC activity of a molecule of interest. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCS) and NK cells. Alternatively, or in addition, the ADCC activity of molecules of interest can be determined as described, for example, by Clynes et al. , 1998, PNAS (USA), 95:652-656.

In this disclosure, the term "halo" when used alone or as part of another group refers to -Cl, -F, -Br, or -I. For example, halo is -Cl or -F.

In this disclosure, the term "hydroxy" when used alone or as part of another group refers to -OH.

In this disclosure, the term "thiol" or the term "sulfhydryl" when used alone or as part of another group refers to -SH.

In this disclosure, the term "alkyl" when used alone or as part of another group refers to an unsubstituted straight or branched aliphatic hydrocarbon containing 1 to 12 carbon atoms, i.e. C _1-12 alkyl, or a specified number of carbon atoms, e.g. C ₁ alkyl such as methyl, C ₂ alkyl such as ethyl, C ₃ alkyl such as propyl or isopropyl, such as methyl, ethyl, propyl, or isopropyl. Refers to C _1-3 alkyl, etc. For example, alkyl is C _1-10 alkyl. In another example, alkyl is C _1-6 alkyl. In another example, alkyl is C _1-4 alkyl. In another example, the alkyl is a straight chain C1-10 alkyl. In another example, alkyl is branched C _3-10 alkyl. In another example, alkyl is straight chain C _1-6 alkyl. In another example, alkyl is branched C _3-6 alkyl. In another example, alkyl is straight chain C _1-4 alkyl. In another example, alkyl is branched C _3-4 alkyl. In another example, alkyl is straight or branched C _3-4 alkyl. Non-limiting exemplary C _1-10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Non-limiting exemplary C _1-4 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, and isobutyl.

In this disclosure, the term "optionally substituted alkyl," when used alone or as part of another group, refers to unsubstituted or independently nitro, hydroxy, cyano , haloalkoxy, aryloxy, alkylthio, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxamide, alkoxycarbonyl, thiol, --N(H)C(=O)NH ₂ , and -- with one, two, or three substituents selected from the group consisting of N(H)C=NH)NH ₂ , optionally substituted aryl, and optionally substituted heteroaryl. Refers to aryl, which is either substituted. For example, an optionally substituted alkyl is substituted with two substituents. In another example, an optionally substituted alkyl is substituted with one substituent. In another example, an optionally substituted alkyl is unsubstituted. Non-limiting exemplary substituted alkyl groups include --CH ₂ OH, --CH ₂ SH, --CH ₂ Ph, --CH ₂ (4-OH)Ph, --CH ₂ (imidazolyl), _{--CH2CH2CO2H , --CH2CH2SO2CH3 , --CH2CH2COPh} , and _{--CH2OC ( =} O _{) CH3 .}

In this disclosure, the term "cycloalkyl" when used alone or as part of another group has, for example, 3 to 12 carbon atoms, containing 1 or 2 double bonds. Refers to an unsubstituted saturated or partially unsaturated cycloaliphatic hydrocarbon containing 1 to 3 rings, ie, C _3-12 cycloalkyl, or having the specified number of carbons. In one example, cycloalkyl has two rings. In another example, cycloalkyl has one ring. In another example, cycloalkyl is saturated. In another example, cycloalkyl is unsaturated. In another example, cycloalkyl is C _3-8 cycloalkyl. In another example, cycloalkyl is C _3-6 cycloalkyl. The term "cycloalkyl" is intended to include groups in which the ring --CH ₂ -- is replaced with --C(=O) --. Non-limiting exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl, cyclohexenyl, cyclopentenyl, and cyclopentanone.

In this disclosure, the term "optionally substituted cycloalkyl" when used alone or as part of another group refers to unsubstituted or independently halo, nitro, Cyano, hydroxy, alkylcarbonyloxy, cycloalkylcarbonyloxy, amino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, Carboxy, carboxyalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted 1, 2, or 3 substituents selected from heterocyclo, alkoxyalkyl, )amino)alkyl, (carboxamido)alkyl, (heterocyclo)alkyl, and --OC(=O)-amino substituted with Refers to cycloalkyl, which is either substituted with The term optionally substituted cycloalkyl refers to a cycloalkyl group having a fused optionally substituted aryl, e.g. phenyl, or a fused optionally substituted heteroaryl, e.g. pyridyl. include. An optionally substituted cycloalkyl having a fused optionally substituted aryl or a fused optionally substituted heteroaryl group is defined as It can be attached to the rest of the molecule. In one example, an optionally substituted cycloalkyl is substituted with two substituents. In another example, an optionally substituted cycloalkyl is substituted with one substituent. In another example, an optionally substituted cycloalkyl is unsubstituted.

In this disclosure, the term "aryl" when used alone or as part of another group refers to an unsubstituted monocyclic or bicyclic aromatic ring system having from 6 to 14 carbon atoms; That is, it refers to a C _6-14 aryl. Non-limiting exemplary aryl groups include phenyl (abbreviated as "Ph"), naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl groups. In one example, the aryl group is phenyl or naphthyl.

In this disclosure, the term "optionally substituted aryl," as used herein alone or as part of another group, refers to , nitro, cyano, hydroxy, thiol, amino, alkylamino, dialkylamino, optionally substituted alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamido, alkyl carbonyl, arylcarbonyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, (cycloalkyl)alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, heterocyclosulfonyl, carboxy, carboxyalkyl, optionally substituted cycloalkyl, alkenyl, Alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxycarbonyl, alkoxyalkyl, (amino)alkyl, (carboxamido)alkyl, and (heterocyclo) Refers to aryl, substituted with any one to five substituents selected from alkyl.

In one example, optionally substituted aryl is optionally substituted phenyl. In another example, an optionally substituted phenyl has 4 substituents. In another example, an optionally substituted phenyl has 3 substituents. In another example, an optionally substituted phenyl has two substituents. In another example, an optionally substituted phenyl has one substituent. In another example, an optionally substituted phenyl is unsubstituted. Non-limiting exemplary substituted aryl groups include 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluoro Phenyl, 3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 2,6-di-fluorophenyl, 2,6-di-chlorophenyl, 2-methyl , 3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3,4-di-methoxyphenyl, 3,5-di-fluorophenyl, 3,5-di-methoxyphenyl, 3,5-dimethoxy, 4- Examples include methylphenyl, 2-fluoro-3-chlorophenyl, 3-chloro-4-fluorophenyl, and 4-(pyridin-4-ylsulfonyl)phenyl. The term optionally substituted aryl includes phenyl groups with fused optionally substituted cycloalkyl or fused optionally substituted heterocyclo groups. An optionally substituted phenyl having a fused optionally substituted cycloalkyl or a fused optionally substituted heterocyclo group is defined as a molecule at any available carbon atom on the phenyl ring. The rest can be attached.

In this disclosure, the term "alkenyl" when used alone or as part of another group refers to alkyl containing one, two, or three carbon-carbon double bonds. In one example, an alkenyl has one carbon-carbon double bond. In another example, alkenyl is C _2-6 alkenyl. In another example, alkenyl is C _2-4 alkenyl. Non-limiting exemplary alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.

In this disclosure, the term "optionally substituted alkenyl," as used herein alone or as part of another group, refers to , nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, From carboxy, carboxyalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, heteroaryl, and optionally substituted heterocyclo Refers to an alkenyl substituted with one, two, or three substituents selected from the group consisting of:

In this disclosure, the term "alkynyl" when used alone or as part of another group refers to an alkyl containing 1 to 3 carbon-carbon triple bonds. In one example, alkynyl has one carbon-carbon triple bond. In another example, alkynyl is C _2-6 alkynyl. In another example, alkynyl is C _2-4 alkynyl. Non-limiting exemplary alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl groups.

In this disclosure, the term "optionally substituted alkynyl," when used herein alone or as part of another group, refers to , nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, selected from the group consisting of carboxy, carboxyalkyl, optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and heterocyclo, Refers to alkynyl, which is substituted with either one, two, or three substituents.

In this disclosure, the term "haloalkyl" when used alone or as part of another group refers to an alkyl substituted with one or more fluorine, chlorine, bromine and/or iodine atoms. In one example, an alkyl group is substituted with 1, 2 or 3 fluorine and/or chlorine atoms. In another example, a haloalkyl group is a C _1-4 haloalkyl group. Non-limiting exemplary haloalkyl groups include fluoromethyl, 2-fluoroethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2 -trifluoroethyl, 3,3,3-trifluoropropyl, 4,4,4-trifluorobutyl, and trichloromethyl groups.

In this disclosure, the term "alkoxy" when used alone or as part of another group refers to an optionally substituted alkyl attached to a terminal oxygen atom, an optionally substituted cycloalkyl, optionally substituted alkenyl, or optionally substituted alkynyl. In one example, alkoxy is an optionally substituted alkyl attached to a terminal oxygen atom. In one example, an alkoxy group is a C _1-6 alkyl attached to a terminal oxygen atom. In another example, an alkoxy group is a C _1-4 alkyl attached to a terminal oxygen atom. Non-limiting exemplary alkoxy groups include methoxy, ethoxy, and tert-butoxy.

In this disclosure, the term "alkylthio" when used alone or as part of another group refers to an optionally substituted alkyl attached to a terminal sulfur atom. In one example, the alkylthio group is a C _1-4 alkylthio group. Non-limiting exemplary alkylthio groups include --SCH ₃ and --SCH ₂ CH ₃ .

In this disclosure, the term "haloalkoxy" when used alone or as part of another group refers to a haloalkyl attached to a terminal oxygen atom. Non-limiting exemplary haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

In this disclosure, the term "heteroaryl" refers to unsubstituted monocyclic and bicyclic aromatic ring systems having from 5 to 14 ring atoms, i.e., 5- to 14-membered heteroaryl; One at least one carbon atom is independently replaced with a heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur. In one example, a heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur. In one example, a heteroaryl has 3 heteroatoms. In another example, a heteroaryl has two heteroatoms. In another example, a heteroaryl has one heteroatom. In another example, a heteroaryl is a 5-10 membered heteroaryl. In another example, a heteroaryl is a 5- or 6-membered heteroaryl. In another example, a heteroaryl is thienyl, which is a 5-membered heteroaryl having 5 ring atoms, such as 4 carbon atoms and 1 sulfur atom. In another example, heteroaryl is pyridyl, which is a 6-membered heteroaryl having 6 ring atoms, eg, 5 carbon atoms and 1 nitrogen atom. Non-limiting exemplary heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuranyl, benzoxazonyl, chromenyl. , xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, sinolinyl, quinazolinyl, pteridinyl, 4aH-carbazolyl , carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, thiazolyl, isothiazolyl, phenothiazolyl, isoxazolyl, furazanyl, and phenoxazinyl. In one example, heteroaryl includes thienyl (e.g., thien-2-yl and thien-3-yl), furyl (e.g., 2-furyl and 3-furyl), pyrrolyl (e.g., 1H-pyrrol-2-yl and 1H -pyrrol-3-yl), imidazolyl (e.g. 2H-imidazol-2-yl and 2H-imidazol-4-yl), pyrazolyl (e.g. 1H-pyrazol-3-yl, 1H-pyrazol-4-yl and 1H -pyridyl (e.g., pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl), pyrimidinyl (e.g., pyrimidin-2-yl, pyrimidin-4-yl, and pyrimidine) -5-yl), thiazolyl (e.g., thiazol-2-yl, thiazol-4-yl, and thiazol-5-yl), isothiazolyl (e.g., isothiazol-3-yl, isothiazol-4-yl, -5 -yl), oxazolyl (e.g., oxazol-2-yl, oxazol-4-yl, and oxazol-5-yl), isoxazolyl (e.g., isoxazol-3-yl, isoxazol-4-yl, and isoxazol-5-yl) ), and indazolyl (eg, 1H-indazol-3-yl). The term "heteroaryl" is also intended to include possible N-oxides. A non-limiting exemplary N-oxide is pyridyl N-oxide.

In one example, a heteroaryl is a 5- or 6-membered heteroaryl. In one example, the heteroaryl is a 5-membered heteroaryl, i.e., the heteroaryl is a monocyclic aromatic ring system having 5 ring atoms, and at least one carbon atom of the ring is independently nitrogen , oxygen, and sulfur. Non-limiting exemplary 5-membered heteroaryl groups include thienyl, furyl, pyrrolyl, oxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, and isoxazolyl. In another example, a heteroaryl is a 6-membered heteroaryl, for example, a heteroaryl is a monocyclic aromatic ring system having 6 ring atoms, and at least one carbon atom of the ring is a nitrogen atom. Replaced. Non-limiting exemplary 6-membered heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, and pyridazinyl.

In this disclosure, the term "optionally substituted heteroaryl" when used alone or as part of another group refers to unsubstituted or independently halo, nitro, Cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl , (cycloalkyl)alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, carboxy, carboxyalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted one, two, selected from aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, (amino)alkyl, (carboxamido)alkyl, and (heterocyclo)alkyl; Refers to a heteroaryl that is substituted with either three or four substituents. In one example, an optionally substituted heteroaryl has one substituent. In another example, an optionally substituted heteroaryl is unsubstituted. Any available carbon or nitrogen atom can be substituted. The term optionally substituted heteroaryl includes heteroaryl groups having fused optionally substituted cycloalkyl or fused optionally substituted heterocyclo groups. An optionally substituted heteroaryl having a fused optionally substituted cycloalkyl or a fused optionally substituted heterocyclo group is defined as It can be attached to the rest of the molecule.

In this disclosure, the term "heterocyclo" when used alone or as part of another group has, for example, 3 to 14 ring members containing 1 or 2 double bonds. , refers to an unsubstituted saturated or partially unsaturated cyclic group containing one, two, or three rings, i.e., 3- to 14-membered heterocyclo, in which at least one carbon atom of one of the rings is Replaced by an atom. Each heteroatom is independently selected from the group consisting of oxygen, sulfur, including sulfoxide and sulfone, and/or nitrogen atoms, which can be oxidized or quaternized. The term "heterocyclo" refers to groups in which the ring --CH ₂ -- is substituted with --C(=O) --, for example, cyclic ureido groups such as 2-imidazolidinone, as well as β-lactams, γ- Contains cyclic amide groups such as lactam, δ-lactam, ε-lactam, and piperazin-2-one. The term "heterocyclo" also includes groups having fused, optionally substituted aryl groups, such as indolinyl or chroman-4-yl. In one embodiment, the heterocyclo group is a C _4-6 heterocyclo, ie, a 4-, 5-, or 6-membered cyclic group containing one ring and one or two oxygen and/or nitrogen atoms. In one embodiment, the heterocyclo group is C _4-6 heterocyclo containing one ring and one nitrogen atom. The heterocyclo may optionally be attached to the remainder of the molecule through any available carbon or nitrogen atom. Non-limiting exemplary heterocyclo groups include azetidinyl, dioxanyl, tetrahydropyranyl, 2-oxopyrrolidin-3-yl, piperazin-2-one, piperazine-2,6-dione, 2-imidazolidinone, piperidinyl. , morpholinyl, piperazinyl, pyrrolidinyl, and indolinyl.

In this disclosure, the term "optionally substituted heterocyclo," as used herein alone or as part of another group, refers to , nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamide, sulfonamide, alkylcarbonyl, cycloalkylcarbonyl, alkoxycarbonyl, _CF3 C(=O)--, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally 1, 2, 3, or 4 selected from heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, (amino)alkyl, (carboxamido)alkyl, or (heterocyclo)alkyl substituted with Refers to heterocyclo, which is either substituted with two substituents. Substitutions may occur on any available carbon or nitrogen atom, or both.

In this disclosure, the term "amino" when used alone or as part of another group refers to a radical of the formula --NRO ^a R ^b , where R ^a and R ^b are each independently selected from the group consisting of hydrogen, optionally substituted alkyl, and aralkyl, or R ^a and R ^b taken together are 3- to 8-membered optionally substituted form a heterocyclo. Non-limiting exemplary amino groups include --NH ₂ and --N(H)(CH ₃ ).

In this disclosure, the term "carboxamide" when used alone or as part of another group refers to a radical of the formula --C(=O)NR ^a R ^b , where R ^a and R ^b is each independently hydrogen, optionally substituted alkyl, hydroxyalkyl, and optionally substituted aryl, optionally substituted heterocyclo, and optionally substituted Heteroaryl or R ^a and R ^b together with the nitrogen to which they are attached form a 3- to 8-membered optionally substituted heterocyclo group. In one embodiment, R ^a and R ^b are each independently hydrogen or optionally substituted alkyl. In one embodiment, R ^a and R ^b together with the nitrogen to which they are attached form a 3-8 membered optionally substituted heterocyclo group. Non-limiting exemplary carboxamide groups include --CONH ₂ , --CON(H)CH ₃ , and --CON(CH ₃ ) ₂ .

In this disclosure, the term "alkoxycarbonyl" when used alone or as part of another group refers to a carbonyl group, ie, --C(=O)-- substituted with alkoxy. In one embodiment, alkoxy is C _1-4 alkoxy. Non-limiting exemplary alkoxycarbonyl groups include --C(=O)OMe, --C(=O)OEt, and --C(=O)OtBu.

In this disclosure, the term "carboxy" when used alone or as part of another group refers to a radical of the formula --CO ₂ H.

In this disclosure, the term "self-immolative group" or "sacrificial group" or "sacrificial linker" refers to all or part of a cleavable linker, which connects two spaced chemical moieties, typically to a stable It is possible to covalently bond to a tripartite molecule, and one of the spaced chemical moieties can be released from the tripartite by enzymatic cleavage, and following enzymatic cleavage, it can be released spontaneously from the rest of the molecule. difunctional chemical moieties that can be cleaved to release the other of the spaced chemical moieties, such as glucocorticosteroids of formula I, II, or III. In some embodiments, the sacrificial linker includes p-aminobenzyl units. In some such embodiments, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate salt is created between the benzyl alcohol and the drug. (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103). In some embodiments, the sacrificial linker is p-aminobenzyloxycarbonyl (PAB). (See Example 3 and Exemplary Embodiments section of this application).

In this disclosure, the term "protecting group" or "PG" is used to block or protect a functionality, e.g., an amine functionality, while a reaction is carried out on another functional group or moiety of the molecule. , refers to the group. Those skilled in the art will be familiar with the selection, attachment, and cleavage of amine protecting groups, many different protecting groups are known in the art, and the suitability of one protecting group or another will depend on the specificity contemplated. It will be understood that it depends on the synthetic scheme. Wuts, P. G. M. ; Greene, T.; W. , “Greene’s Protective Groups in Organic Synthesis”, 4th Ed. , J. Papers on this subject are available for reference, such as Wiley & Sons, NY, 2007. Suitable protecting groups include carbobenzyloxy (Cbz), tert-butyloxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), and benzyl (Bn) groups. In one embodiment, the protecting group is a BOC group.

In this disclosure, the term "ethylene glycol" refers to a chemical with the formula -OCH2CH2O-.

In this disclosure, the term "ethylene oxide" refers to a chemical of the formula -CH2CH2O-.

As used in this disclosure and the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

Whenever an embodiment is described herein with the term "comprising," an otherwise similar embodiment described with the term "consisting of" and/or "consisting essentially of" It is understood that configurations are also provided.

As used herein in phrases such as "A and/or B," the term "and/or" refers to both "A and B," "A or B," "A," and "B." intended to include. Similarly, the term "and/or" used in phrases such as "A, B, and/or C" is intended to include each of the following embodiments: A, B, and C. A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, "autoimmunity" or "autoimmune disease or condition" means a disease or disorder arising from and directed against an individual's own tissues or co-isolates or expressions thereof; Broadly refers to and includes conditions. As used herein, autoimmune conditions include inflammatory or allergic conditions, e.g., host cells against autoantigens characterized by inflammation and/or potentially associated with tissue destruction, such as rheumatoid arthritis, for which steroids are an effective treatment. Includes chronic diseases characterized by an immune response.

An "allergic disease or condition" or "allergic reaction" is a condition caused by hypersensitivity of the immune system to typically harmless substances or antigens in the environment. These diseases include, by way of example, atopic dermatitis, allergic asthma, primary immunodeficiency, chronic sinusitis, eosinophil-related diseases, and other conditions involving allergic responses or reactions.

"Immune cell" as used herein broadly refers to cells of hematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes such as B cells and T cells, natural killer cells, dendritic cells, as well as monocytes, macrophages, eosinophils, mast cells, basophils, and other immune cell types. These include, but are not limited to, bone marrow cells such as granulocytes.

As used herein, "immune-related disease (or disorder or condition)" refers to acute and chronic organ transplant rejection, allogeneic stem cell transplantation, autologous stem cell transplantation, bone marrow transplantation, and transplantation, including graft-versus-host disease. It should be understood to include any disease, disorder, or condition selected from the group including, but not limited to, autoimmune diseases, inflammatory disorders, and immune disorders associated with graft rejection.

"Inflammatory disorder," "inflammatory condition," and/or "inflammation," used interchangeably herein, broadly refer to chronic or acute inflammatory diseases, including inflammatory autoimmune diseases and inflammatory allergic conditions. explicitly included. These conditions include, by way of example, inflammatory disorders characterized by a dysregulated immune response to noxious stimuli such as pathogens, damaged cells, or irritants. Inflammatory diseases underlie a variety of human diseases. Non-immune diseases with an etiology in inflammatory processes include cancer, atherosclerosis, and ischemic heart disease. Examples of inflammation-related disorders include chronic prostatitis, glomerulonephritis, hypersensitivity, pelvic inflammatory disease, reperfusion injury, sarcoidosis, vasculitis, interstitial cystitis, and orthocomplementemic urticaria. vasculitis, pericarditis, myositis, antisynthetic enzyme syndrome, scleritis, macrophage activation syndrome, Behcet's syndrome, PAPA syndrome, Blau syndrome, gout, adult and juvenile Still's disease, cryopyrin-associated periodic fever syndrome, Muckle-Wells syndrome, familial cold-induced autoinflammatory syndrome, neonatal onset multisystem inflammatory disease, familial Mediterranean fever, chronic infantile neurological, skin and joint syndrome, systemic juvenile idiopathic arthritis, hyper-IgD syndrome, Schnitzler syndrome, TNF receptor-associated periodic syndrome (TRAPSP), gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty arthritis, as well as psoriasis, atopic dermatitis, eczema, rosacea inflammatory skin disorders selected from , urticaria, and acne.

As used herein, "mammal" refers to any warm-blooded vertebrate mammal, including humans, characterized by a coat of hair on the skin and, in females, mammary glands that produce milk to feed their young. Refer broadly. Examples of mammals include alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cows, dogs, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, Includes, but is not limited to, sheep, shrews, squirrels, tapirs, and voles. Mammals include, but are not limited to, bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammals include Washington D.C. C. Also included are any and all listed in the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington, D.C.

"Patient," or "subject," or "recipient," "individual," or "individual treated" are used interchangeably herein and are used interchangeably to treat the symptoms of a medical condition, to alleviate a medical condition, or to treat the occurrence or relapse of a medical condition. Broadly refers to any animal that requires treatment in any way to prevent it. "Patient" as used herein also refers to having risk factors, medical history, susceptibility, symptoms, and signs of, having been previously diagnosed with, at risk for, or developing a disease. Broadly refers to any animal that is a member of the patient population. The patient can be a clinical patient, such as a human, or a veterinary patient, such as a pet, farm animal, livestock, exotic animal, or zoo animal.

A "subject" or "patient" or "individual" in the context of therapy or diagnosis herein includes any human or non-human animal. The term "non-human animal" includes all vertebrates, including mammals and non-mammals such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Any animal suitable for treatment according to the invention includes, but is not limited to, avian and mammalian subjects, preferably mammals. Any mammalian subject in need of being treated according to the present invention is suitable. Human subjects of both genders and any stage of development (ie, neonates, infants, young adults, adolescents, and adults) can be treated according to the present invention. The invention may also be practiced on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, cows, goats, sheep, and horses, for veterinary purposes, and for drug screening and drug development purposes. obtain. "Subject" is used interchangeably with "individual" and "patient."

"Therapy," "therapeutic," "treating," or "treatment" as used herein refers to treating a disease, preventing or reducing the progression of a disease or its clinical symptoms, and Broadly refers to alleviating a disease and/or causing regression of the disease or its clinical symptoms. Therapy includes the prevention, treatment, remedy, reduction, alleviation, and/or provision of relief from a disease, sign, and/or symptom of a disease. Therapy includes the alleviation of signs and/or symptoms of disease (eg, inflammation, pain) in patients who are developing them. Therapy also encompasses "prophylaxis." The term "reduced" for purposes of therapy broadly refers to a clinically significant reduction in signs and/or symptoms. Therapy includes treatment of recurrence or recurrent signs and/or symptoms (eg, inflammation, pain). Therapy includes, but is not limited to, preventing the development of signs and/or symptoms at any time, as well as reducing existing signs and/or symptoms and eliminating existing signs and/or symptoms. Therapy includes treatment of chronic diseases ("maintenance") and acute diseases. For example, treatment includes treatment or prevention of recurrence or recurrence of signs and/or symptoms (eg, inflammation, pain).

After defining certain terms and phrases used in this application, novel glucocorticosteroid agonists, glucocorticosteroid agonist linkers, and ADCs containing the same, methods for their production and use according to the present invention will be described. , further described below.

The present invention provides for linkers to antibodies or antibody fragments that contain an antigen binding region that binds to an immune cell antigen, typically a human immune cell antigen, such as human V domain Ig inhibitor of T cell activation (VISTA). ADCs comprising a novel glucocorticosteroid agonist of formula I, II, or III attached directly or indirectly to an ADC. However, we demonstrate herein that glucocorticosteroid agonists and ADCs comprising glucocorticosteroid agonist linkers according to the present invention are also effective when conjugated to antibodies or fragments that bind to immune cell antigens other than VISTA. It is shown.

In some exemplary embodiments, the ADC comprises an antibody or fragment that has a short serum half-life under physiological pH conditions (pH 7.5), such as an antibody or fragment in rodents (human VISTA knock-in). The serum half-life of is generally 1-72 hours, 1-32 hours, 1-16 hours, 1-8 hours, 1-8 hours in human VISTA knock-in rodents at physiological pH conditions (pH 7.5). 4 hours, or 1-2 hours ± 0.5 hours, or in primates (cynomolgus monkeys) ≈3.5, 3, 2.5, or 2.3 days ± 0.5 days, and the anti-human VISTA antibody or the antibody fragment contains an anti-inflammatory agent, such as a steroid or corticosteroid receptor agonist of Formula I, II, or III, or a corticosteroid receptor agonist linker containing the same as disclosed herein. , or a functional derivative or radical thereof, i.e., when administered to a subject, e.g., a human or other mammal, when released from an ADC containing it, produces the desired anti-inflammatory properties upon internalization into immune cells. It is attached directly or indirectly through a linker to the derivative that induces the inflammatory effect.

In particular, in the case of an ADC comprising an anti-VISTA antibody or antibody fragment, the ADC specifically binds to VISTA-expressing immune cells at physiological pH, and the corticosteroid receptor agonist of formula I, II, or III is a neutrophil. from ADCs upon internalization into target (immune) cells such as monocytes, macrophages, T cells, CD4 T cells, CD8 T cells, Tregs, and other immune cells present in the peripheral blood. will be released. This release of corticosteroid receptor agonists is apparently triggered by enzymes, such as esterases, that provide cleavage of the ADC after it has been internalized by target immune cells. Release of the corticosteroid receptor agonist from the contained ADC upon internalization into immune cells then selectively exerts the desired anti-inflammatory effect within immune cells expressing the antigen bound by the ADC, e.g., VISTA. induce. As mentioned above, the effectiveness of corticosteroid receptor agonists (anti-inflammatory activity) is due to the fact that such steroid compounds are internalized by cells, e.g., immune cells expressing VISTA or other antigens bound by ADCs. It is achieved only after

In a preferred embodiment, the antibody or fragment, e.g., an anti-VISTA antibody or fragment, is silent, i.e., has an Fc region mutated to impair FcR binding, e.g., a silent IgG1, IgG2, IgG3, or IgG4, most typically may include silent IgG2 or silent IgG1, or the antibody or fragment may lack an Fc region or include an Fc fragment that does not bind FcR. Exemplary silent Fc regions are disclosed below. Thus, in some cases, an ADC comprising an antibody or fragment, e.g., an anti-VISTA antibody or fragment, that binds to an antigen-expressing immune cell and binds to an immune cell antigen while internalized therein, thereby It will not induce a modulatory effect on the bound antigen, eg, VISTA, ie, it will not agonize or antagonize the effects of the antigen to which it binds, eg, it will not agonize or antagonize the suppressive effects of VISTA on immunity. Rather, the therapeutic effect elicited by the ADC is due solely or primarily to the anti-inflammatory agent(s) bound to it, i.e., the corticosteroid receptor agonist of formula I, II, or III; When included in an ADC, it is internalized and released into immune cells upon administration, inducing the desired anti-inflammatory effect exclusively or preferentially in the target immune cells expressing the antigen bound by the ADC. .

Because the subject ADCs, e.g., anti-VISTA ADCs, selectively bind to target immune cells, e.g., myeloid cells, T cells, neutrophils, monocytes, etc., the subject ADCs are potent in many immune cells. However, many anti-inflammatory drugs, such as corticosteroid receptor agonists such as dexamethasone, budesonide, and other steroids, can still be internalized by non-target cells by such steroid compounds. It will alleviate or prevent the harmful side effects induced.

Additionally, the subject ADCs, e.g., naive and activated target VISTA-expressing immune cells, e.g., naive and activated monocytes, macrophages, T cells, T regs, CD4 T cells, CD8 T cells, neutrophils, eosinophils. Anti-VISTA ADCs that selectively bind to and internalize dendritic cells, NK cells, and bone marrow cells include dexamethasone, budesonide, and others previously identified and commonly known in the art. Compared to conventional free steroids such as steroids, the use of reduced doses of the corticosteroid receptor agonists of the present invention may be facilitated. Also, the subject corticosteroid receptor agonist compounds of Formula I, II, or III, or containing corticosteroid receptor agonist linker compounds, are capable of inhibiting the use of antibodies that target other immune cell antigens. Any or all of these specific types of immune cells expressing such antigens can be used to treat conditions where they are involved in disease pathology.

In the specific case of a VISTA ADC comprising a subject corticosteroid receptor agonist compound of Formula I, II, or III, or containing a corticosteroid receptor agonist linker compound, the subject ADC contains an anti-inflammatory agent. Previously reported ADCs to target and induce internalization, particularly those targeting steroid internalization into immune cells, such as ADCs targeting CD74, CD163, TNF, and PRLR possesses a unique combination of advantages compared to cells but still induce prolonged PD), as well as the advantages of the novel subject corticosteroid receptor agonist compounds of Formula I, II, or III provided herein.

In particular, in the specific case of VISTA ADCs, the subject ADCs bind VISTA-expressing immune cells at very high densities and remain effective therein for long periods despite their very short PK. (inducing anti-inflammatory activity) and are therefore well suited for treating chronic inflammatory or autoimmune or allergic diseases where long-term and repeated administration of steroids is therapeutically warranted.

In the specific case of VISTA ADCs, the subject ADCs also target a wide range of immune cells, including neutrophils, bone marrow, T cells, Tregs, macrophages, and endothelial cells, or allergy, inflammation, and autologous ADCs that bind to other antigens expressed on immune cells involved in immune responses and conditions, and thus the subject ADCs are associated with diseases such as inflammatory or autoimmune or allergic diseases, as well as these types of immune cells. It can be used to treat conditions associated with inflammation, such as heart disease, ARDS, cancer, and infections involving any or all of the following. For example, the subject ADCs can be used to treat or prevent inflammation associated with bacterial or viral infections, such as COVID-19, influenza virus, pneumonia (viral or bacterial) infections, and the like. However, when the novel glucocorticosteroid steroid agonist linkers of formulas (I), (II), and (III) provided herein are coupled to antigens that target other immunizing antigens, The present invention is not limited to VISTA ADCs as Applicants have shown that they are effectively internalized and release the active steroid payload therein.

Furthermore, the subject ADCs have a rapid onset of efficacy and can, for example, induce anti-inflammatory activity within 2 hours after administration, and thus can be used for acute treatment, which can be rapidly treated. Such as COVID-19 and other coronaviruses, influenza viruses, pneumonia (viral or bacterial) infections, and the like, which could otherwise result in cytokine storm, ARDS, and in worst cases sepsis or septic shock. may be particularly useful in the context of treating/preventing inflammation associated with bacterial or viral infections.

Furthermore, in the specific case of anti-VISTA ADCs, VISTA, unlike some other ADC target antigens, is expressed exclusively by immune cells, and thus the ADC of interest internalizes non-target cells. The tendency disappears.

Also, in the specific case of anti-VISTA ADCs, the subject ADCs should not bind to B cells, but should not be as immunosuppressive as free steroids, which could lead to repeated and/or prolonged use of the subject ADCs. This is an unintended consequence of long-term immunosuppression due to long-term steroid use, as chronic steroid use has been correlated with several cancers, infections, and other conditions. Probability is high. However, when the ADCs of the present invention are bound to antibodies that target other antigens, these ADCs may cause disease in any or all of these specific types of immune cells to which they are bound. It is to be understood that it may be used to treat conditions involving pathology.

Additionally, in the specific case of a VISTA ADC comprising a subject corticosteroid receptor agonist compound of Formula I, II, or III, or a corticosteroid receptor agonist linker compound containing the same, the subject ADC is , act on Tregs, key immune cells involved in steroid efficacy, and therefore, particularly with respect to previous ADCs containing corticosteroid receptor agonist compounds, autoimmune, allergic, or inflammatory conditions, or inflammation involving Tregs. may be broadly or specifically more effective in treating.

Additionally, in the specific case of a VISTA ADC comprising a subject corticosteroid receptor agonist compound of Formula I, II, or III, or a corticosteroid receptor agonist linker compound containing the same, the subject ADC is: Both resting (naïve) and activated immune cells, such as monocytes, macrophages, T cells, T regs, CD4 T cells, CD8 T cells, neutrophils, eosinophils, dendritic cells, NK cells, and acting on bone marrow cells (where VISTA is constitutively expressed), and as a result, the subject ADCs will remain active during both active and remission phases of allergic, inflammatory, and autoimmune conditions ( induces anti-inflammatory activity).

Additionally, because the VISTA ADCs, including the subject corticosteroid receptor agonist compounds of formula I, II, or III, act on neutrophils, and that immune cell is important in acute inflammation, the subject ADCs may It may be useful in treating inflammatory or autoimmune or allergic conditions characterized by inflammation and/or rare or sporadic inflammatory attacks.

Additionally, the subject ADCs comprising novel glucocorticosteroid steroid agonist linkers of formula (I), (II) or (III) advantageously rapidly internalize immune cells and deliver large amounts of active steroid payload. and provide rapid and long-term effectiveness.

In the specific case of VISTA ADCs, ADCs have been shown to internalize immune cells very quickly (e.g., within about 30 minutes) due to high VISTA cell surface turnover, which It is further shown that the subject ADCs are well suited for treating inflammatory or autoimmune or allergic conditions characterized by acute inflammation and/or infrequent or sporadic inflammatory attacks.

Additionally, in some cases, the subject ADC has a very short half-life (PK) and binds only to immune cells, and therefore the subject ADC has a very short half-life (PK), such as Humira Compared to other ADCs containing antibodies, it should be less prone to target-related toxicity and undesired peripheral steroid exposure (lower non-specific loss effects).

Furthermore, in some embodiments, the biological activity (anti-inflammatory effect) of the subject ADC is entirely due to the anti-inflammatory payload (steroid) contained therein, i.e., the antibody, e.g., an anti-VISTA antibody, In cases with silent IgGs, such as silent IgG1 or IgG2 Fc regions, they do not induce VISTA-mediated immunological functions (do not block any VISTA biology).

Based on at least the combination of the foregoing advantages, the subject ADCs are well suited for acute and chronic use, and for both therapeutic and prophylactic use, i.e., reducing or inhibiting inflammation, preventing the development of inflammation, It would be suitable for use in prolonging the inactive phase of a disease, as well as in the treatment of a myriad of different types of inflammatory, allergic and autoimmune diseases.

As mentioned above, in some embodiments, the subject ADCs are anti-VISTA antibodies that bind to VISTA (generally human VISTA) expressing immune cells at physiological pH conditions and possess a short half-life or PK. including. Typically, these antibodies contain a silent Fc or no Fc, and binding of the ADC to VISTA-expressing cells does not induce any effect on VISTA signaling or VISTA-mediated effects on immunity. Dew.

In contrast, in some embodiments, the antibody in the ADC, e.g., an anti-VISTA antibody or an antibody targeting another immune cell antigen, is a functional IgG, e.g., functional IgG1, IgG2, IgG3, or IgG4. will include. In the case of anti-VISTA antibodies that contain functional IgG2, such ADCs may inhibit VISTA or other immune cell antigen-mediated signaling or VISTA or other immune cell antigen-related functions, such as T cell proliferation and T cell activation. suppression, as well as the suppression of some inflammatory cytokines. This may result in additive or synergistic effects on suppressing inflammation, allergic reactions, and/or autoimmunity.

CDR and variable sequences of exemplary anti-VISTA antibodies and antibody fragments, i.e., those possessing fragments with short serum half-lives under physiological pH conditions (≈pH 7.5), such as antibodies in cynomolgus monkeys or humans or The serum half-life of the fragment is generally about 2.3 days ± 0.7 days or less under physiological pH conditions (≈pH 7.5), and in rodents (human VISTA knock-in), it is generally , 1-72 hours, 1-32 hours, 1-16 hours, 1-8 hours, 1-4 hours, or 1-2 hours ± 0.5 hours in human VISTA knock-in rodents, or in primates (cynomolgus monkeys) ≈3.5, 3, 2.5, or 2.3 days ± 0.5 days can be found in FIGS. 8, 10, and 12.

Exemplary inflammatory agents that can be incorporated into the ADCs of the invention, i.e., conjugated to anti-VISTA antibodies and anti-VISTA antibody fragments, e.g., via a linker, optionally further by a heterobifunctional group. steroids or corticosteroid receptor agonists such as those previously described generally, more specifically budesonide, beclomethasone, betamethasone, cicledonide, cortisol, cortisone, cortisone acetate, 16-alpha hydroxyprednisolone, Dexamethasone, difluorazone, etamethasoneb, flumethasone, flunisolide, fluocinone acetonide, fludrocortisone, fluticasone propionate (Flovent(TM), Flonase(TM)), hydrocortisone, ciclesonide, methylprednisolone, prednisone, prednisolone, mometasone, Pulmicort, triamcinolone, triamcinolone acetonide, or another steroid compound or derivative thereof possessing anti-inflammatory or steroid activity, in particular the novel steroids and steroid linkers of formula I, II, or III according to the invention, and their functional Contains derivatives. Preferred exemplary ADCs, steroids, and steroid linkers according to the invention are disclosed in the Examples below, particularly Example 3 in the Exemplary Embodiments section, and illustrated in FIGS. 118A-O.

The subject ADCs may be used to treat a subject, e.g., a human or non-human mammal, with any condition for which alleviation of inflammation is therapeutically warranted by the use of anti-inflammatory agents such as steroids. is planned. Such conditions may be associated with acute or chronic inflammation, such as sporadic or paroxysmal inflammation. In some preferred embodiments, the subject has a condition requiring repeated and/or high doses of an anti-inflammatory agent, such as a corticosteroid receptor agonist, and under conventional conditions, i.e. Administering the drug when it is naked or unconjugated can induce undesirable side effects such as toxicity to non-target cells. Such conditions include autoimmune and inflammatory conditions. Non-limiting examples of such conditions include allergies, autoimmunity, transplantation, gene therapy, inflammation, GVHD or sepsis, infections, cancer, or related to any of the aforementioned conditions in a human subject. This includes treating or curing inflammatory, autoimmune, or allergic side effects.

In some other preferred embodiments, the subject has an acute or chronic inflammatory condition or flare-ups, for example, characterized by frequent or infrequent repeated acute inflammatory attacks, for which a rapid onset of efficacy is therapeutically desirable. Inflammatory conditions, optionally conditions for which repeated and/or high doses of anti-inflammatory agents such as corticosteroid receptor agonists are therapeutically warranted, optionally under conventional conditions, i.e. If the agent is naked or unconjugated, there will be situations where administering the drug may induce undesirable side effects, such as toxicity to non-target cells. Such conditions include, for example, autoimmune and inflammatory conditions characterized by acute and/or severe inflammatory attacks, cancer, and infectious conditions associated with inflammation.

Non-limiting examples of such conditions include allergies, autoimmunity, transplantation, gene therapy, inflammation, cancer, GVHD or sepsis, infections (e.g., bacterial, viral, fungal, parasitic), acute respiratory distress. syndrome (ARDS), or inflammatory, autoimmune, or allergic side effects associated with any of the aforementioned conditions in a human subject.

Other specific exemplary conditions in which use of the subject ADCs may be beneficial include rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcers. including genital colitis, plaque psoriasis, sweat gland abscess, uveitis, Behcet's disease, spondyloarthropathies, or psoriasis.

Other exemplary conditions and instances where use of the subject ADCs may be therapeutically beneficial include:
(i) a condition that can be effectively treated only primarily with high doses of steroids, optionally where the patient is being treated with or is being treated with high doses of steroids; polymyalgia and/or giant cell arteritis,
(ii) Conditions with comorbidities that limit steroid use, optionally diabetes, nonalcoholic steatohepatitis (NASH), morbid obesity, avascular necrosis/osteonecrosis (AVN), glaucoma, steroid-induced high blood pressure, severe skin fragility, and/or osteoarthritis;
(iii) conditions for which safe long-term therapeutic agents are available, but where induction for several months with high doses of steroids is desired; optionally, induction for several months with high doses of steroids is therapeutic; AAV, polymyositis, dermatomyositis, lupus, inflammatory lung disease, autoimmune hepatitis, inflammatory bowel disease, immune thrombocytopenia, autoimmune hemolytic anemia, gout patients,
(iv) skin conditions requiring short-term/long-term treatment, optionally conditions of uncertain treatment or duration, and/or conditions for which there are no effective alternatives to steroid administration, optionally Stevens; Johnson, other severe drug eruption conditions, conditions with extensive contact dermatitis, other severe immune-related skin conditions such as PG, LCV, erythroderma, and the like;
(v) conditions treated with high-dose corticosteroids for flare-ups/relapses, optionally COPD, asthma, lupus, gout, pseudogout;
(vi) immune-related neurological diseases such as small fiber neuropathy, MS (subset), chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, and the like;
(vii) optionally, hematology/oncology indications for which high doses of steroids are therapeutically warranted or beneficial;
(viii) ophthalmological conditions, optionally uveitis, iritis, scleritis, and the like;
(ix) conditions associated with persistent or very long-term adrenal insufficiency or secondary adrenal insufficiency, optionally an iatrogenic Addison's disease crisis;
(x) Conditions often treated with long-term, low-dose steroids, optionally lupus, RA, psA, vasculitis, and the like; and (xi) Pregnant/lactating women, pediatric patients, optionally Selectively, special classes of patients such as pediatric patients with growth defects or cataracts.

A composition containing an ADC of formula I, II, or II according to the invention or a novel glucocorticosteroid, or a steroid linker containing it, alone or with other therapeutic agents, in particular autoimmune, allergic, or other immunosuppressive molecules or anti-inflammatory agents or other therapeutic agents used in treating inflammatory conditions, such as acquired immunodeficiency syndrome (AIDS), acquired splenic atrophy, acute anterior uveitis, Acute disseminated encephalomyelitis (ADEM), acute gouty arthritis, acute necrotizing hemorrhagic leukoencephalitis, acute or chronic sinusitis, acute suppurative meningitis (or other central nervous system inflammatory disorders), acute severe Inflammation, Addison's disease, adrenalitis, adult-onset diabetes mellitus (type II diabetes), adult-onset idiopathic hypoparathyroidism (AOIH), agammaglobulinemia, agranulocytosis, vasculitis (vasculitis, optionally macroangiitis, optionally polymyalgia rheumatica, and giant cell (Takayasu) arthritis), allergic conditions, allergic contact dermatitis, allergic dermatitis, allergic Granulomatous vasculitis, allergic hypersensitivity disorder, allergic neuritis, allergic reaction, alopecia areata, alopecia totalis, Alport syndrome, alveolitis, optionally allergic alveolitis or fibrotic lung cystitis, Alzheimer's disease, amyloidosis, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), eosinophil-related disorders, optionally eosinophilia, anaphylaxis, ankylosing spondylitis, vasodilation disease, antibody-mediated nephritis, anti-GBM/anti-TBM nephritis, antigen-antibody complex-mediated disease, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, antiphospholipid syndrome (APS), aphthous, aphthous stomatitis, regeneration delinquent anemia, arrhythmia, arteriosclerosis, arteriosclerotic disorders, arthritis, optionally rheumatoid arthritis, such as acute or chronic rheumatoid arthritis, chronic progressive arthritis, osteoarthritis, ascariasis, aspergilloma, granulomas containing eosinophils, aspergillosis, azoospermia, asthma, optionally asthma bronchiale, bronchial asthma, or autoimmune asthma, ataxia telangiectasia, Ataxic sclerosis, atherosclerosis, autism, autoimmune angioedema, autoimmune aplastic anemia, autoimmune atrophic gastritis, autoimmune blood diabetes, autoimmune orchitis and ovaries autoimmune disorders, including autoimmune disorders of the testis and ovaries, including inflammation of the testis, autoimmune disorders associated with collagen diseases, autoimmune autonomic neuropathy, autoimmune ear disease (AGED), and thyroiditis, such as autoimmune thyroiditis; Endocrine diseases, autoimmune enteropathy syndrome, autoimmune hypogonadism, autoimmune hearing loss, autoimmune hemolysis, autoimmune hepatitis, autoimmune liver damage, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune neutropenia, autoimmune pancreatitis, autoimmune polyendocrinopathy, polyglandular autoimmune syndrome type I, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, autoimmune-mediated gastrointestinal disease, axonal and neuronal neuropathy, Baro's disease, Behcet's disease, Benign familial and ischemia-reperfusion injury, benign lymphocytic vasculitis, Buerger's disease (IgA nephropathy), avian pulmonary disease, blindness, Beck's disease, bronchiolitis obliterans (non-transplant) vs. NSIP, bronchitis, bronchus Pneumonic aspergillosis, Breton syndrome, bullous pemphigoid, Kaplan syndrome, cardiomyopathy, cardiovascular ischemia, Castleman syndrome, celiac disease, celiac sprue (gluten enteropathy), brain degeneration, cerebral ischemia, and blood vessels. Diseases associated with neoplasia, Chagas disease, channelopathies, optionally epilepsy, channelopathies of the CNS, chorioretinitis, choroiditis, autoimmune blood disorders, chronic active hepatitis or autoimmune chronic active hepatitis, chronic Contact dermatitis, chronic eosinophilic pneumonia, chronic fatigue syndrome, chronic hepatitis, chronic hypersensitivity pneumonitis, chronic inflammatory arthritis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic refractory inflammation, chronic mucocutaneous candida chronic neurological disorder, optionally IgM polyneuropathy or IgM-mediated neuropathy, chronic obstructive airway disease, chronic pulmonary inflammatory disease, chronic relapsing multifocal osteomyelitis (CRMO), chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, coronavirus-mediated infections such as SARS-CoV-2 (COVID-19), SARS- CoV, MERS, SARS-CoV-2 and related side effects, CNS inflammatory disorders, CNS vasculitis, celiac disease, Cogan syndrome, cold agglutinin disease, polypoid colitis, colitis, e.g. colitis), ulcerative colitis (colitis ulcerosa), collagen fibrous colitis, conditions with T cell infiltration and chronic inflammatory response, congenital heart block, congenital rubella infection, Coombs positive anemia, coronary artery disease, Coxsackie myocardium CREST syndrome (calcinosis, Raynaud's phenomenon), Crohn's disease, cryoglobulinemia, Cushing's syndrome, cyclitis, optionally chronic cyclitis, heterochronic cyclitis , iridocyclitis or Fuchs' cystitis, cystic fibrosis, cytokine-induced toxicity, hearing loss, degenerative arthritis, demyelinating diseases, optionally autoimmune demyelinating diseases, demyelinating nerves Dengue fever, dermatitis including dermatitis herpetiformis and atopic dermatitis, contact dermatitis, dermatomyositis, skin diseases with acute inflammatory components, Devic's disease (neuromyelitis optica), diabetic aortopathy, diabetic kidney disease diabetic retinopathy, Diamond-Blackfan anemia, diffuse interstitial pulmonary fibrosis, dilated cardiomyopathy, discoid lupus, diseases with leukocyte leakage, Dressler syndrome, Dupuytren's contracture, echovirus infection , eczema including allergic or atopic eczema, encephalitis such as Rasmussen's encephalitis and limbic encephalitis and/or brainstem encephalitis, encephalomyelitis, optionally allergic encephalomyelitis or allergic encephalomyelitis. encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), intraarterial hyperplasia, endocarditis, endocrine ophthalmopathy, endometriosis, endomyocardial fibrosis, enophthalmos lens hypersensitivity Endophthalmitis, endophthalmitis, allergic enteritis, eosinophilia/myalgia syndrome, eosinophilic fasciitis, epidemic keratoconjunctivitis, epidermolysis bullosa acquired (EBA), episclera, episclera inflammation, Epstein-Barr virus infection, persistent erythema protuberans, erythema multiforme, erythema nodosum, erythema nodosum, erythroblastosis fetalis, esophageal motility disorder, essential mixed cryoglobulinemia, ethmoid bone , Evan syndrome, experimental allergic encephalomyelitis (EAE), factor VIII deficiency, farmer's lung disease, rheumatic fever (febris rheumatica), Felty syndrome, fibromyalgia, fibrosing alveolitis, filariasis, nest Segmental glomerulosclerosis (FSGS), food poisoning, anterior gastric atrophy, giant cell arthritis (temporal arthritis), giant cell hepatitis, giant cell polymyalgia, glomerulonephritis, chronic or acute glomerulonephritis ( glomerulonephritis (GN) with and without nephrotic syndrome (e.g., primary GN), Goodpasture syndrome, gouty arthritis, granulocyte transfusion-associated syndrome, granulomatosis including lymphomatoid granulomatosis, polyvascular Inflammatory granulomatosis (GPA), granulomatous uveitis, Graves' disease, Guillain-Barre syndrome, guttate psoriasis, hemoglobinuria attacks, Hammann-Rich disease, Hashimoto's disease, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemochroma tosis, immune-mediated hemolytic anemia (AIHA), including hemolytic anemia or autoimmune hemolytic anemia, hemolytic anemia, hemophilia A, Henoch-Schönlein purpura, herpes gestationis, human immunodeficiency virus (HIV) Infection, hyperalgesia, hypogammaglobulinemia, hypogonadism, hypoparathyroidism, idiopathic diabetes insipidus, idiopathic facial paralysis, paralysis, idiopathic hypothyroidism, idiopathic IgA nephropathy, sudden onset membranous GN or idiopathic membranous nephropathy, idiopathic nephritic syndrome, idiopathic pulmonary fibrosis, idiopathic sprue, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgE-mediated disease, optional anaphylaxis and allergic or atopic rhinitis, IgG4-associated sclerosing disease, localized ileitis, immune complex nephritis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, immune-mediated GN, immunomodulatory lipoproteins including adult or acute respiratory distress syndrome (ARDS), inclusion body myositis, infectious arthritis, infertility due to anti-sperm antibodies, inflammation of all or part of the uvea, inflammatory bowel disease. (IBD) Inflammatory hyperproliferative skin disease, inflammatory myopathy, insulin-dependent diabetes (type 1), insulitis, interstitial cystitis, interstitial lung disease, interstitial pulmonary fibrosis, iritis, Blood reperfusion injury, arthritis, juvenile arthritis, juvenile dermatomyositis, juvenile diabetes, juvenile onset (type I) diabetes mellitus, childhood insulin-dependent diabetes mellitus (IDDM), juvenile rheumatoid arthritis, Kawasaki syndrome, horn sicca Conjunctivitis, kypanosomiasis, Lambert-Eaton syndrome, leishmaniasis, leprosy, leucopenia, leukocyte adhesion deficiency, leukocytoclastic vasculitis, leukopenia, lichen planus, lichen sclerosus scabies, ligneous conjunctivitis, linear IgA dermatopathy, linear IgA disease (LAD), Leffler syndrome, lupoid hepatitis, lupus (including nephritis, encephalitis, pediatric, non-renal, extrarenal, discoid, alopecia), Lupus (SLE), disseminated lupus erythematosus, Lyme arthritis, Lyme disease, lymphocytic interstitial pneumonia, malaria, male and female autoimmune infertility, upper jaw, medium-sized vasculitis (Kawasaki disease and polyarteritis nodosa) membrano- or membranous proliferative GN (MPGN), including types I and II, and rapidly progressive GN, membranous nephropathy, Ménière's disease, inflammation, microscopic colitis, microscopic polyangiitis, migraine, minimal change nephropathy, mixed connective tissue disease (MCTD), infectious mononucleosis, Moren's ulcer, Mucha-Habermann disease, multifocal Motor neuropathy, multiple endocrine insufficiency, multiple organ failure syndromes (such as those secondary to sepsis, trauma, or hemorrhage), multiple organ failure syndromes, multiple sclerosis (MS), including spino-visual MS, and multiple sclerosis. myasthenia gravis, myasthenia gravis, myocarditis, myositis, narcolepsy, necrotizing enterocolitis, and full-thickness colitis, and autoimmune inflammation. Genital intestinal disease, necrotizing, cutaneous, or hypersensitivity vasculitis, neonatal lupus syndrome (NLE), nephrosis, nephrotic syndrome, neurological disease, neuromyelitis optica (Devick's disease), neuromyelitis optica, neuromyotonia, neutropenia , non-cancerous lymphocytosis, non-granulomatous uveitis, non-malignant thymoma, ocular and orbital inflammatory disorders, ocular cicatricial pemphigoid, ovariitis, sympathetic ophthalmitis, opsoclonus-myoclonus syndrome ( OMS), opsoclonus myoclonic syndrome (OMS), and sensory neuropathy, optic neuritis, granulomatous orchitis, osteoarthritis, relapsing rheumatoid arthritis, pancreatitis, pancytopenia, PANDAS (pediatric autoimmunity associated with Streptococcus neuropsychiatric disorders), paraneoplastic syndromes including paraneoplastic cerebellar degeneration, paraneoplastic syndromes, paraneoplastic neurological syndromes, optionally Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, parasitic diseases such as Leishmania , paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, parsonage-Turner syndrome, Parsonage-Turner syndrome, parvovirus infection, bullous pemphigoid and cutaneous pemphigoid. Pemphigus, pemphigus (including pemphigus vulgaris), pemphigus erythematous, pemphigus foliaceus, pemphigus mucosa pemphigoid, pemphigus, peptic ulcer, periodic quadriplegia, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (anemia perniciosa), pernicious anemia, lens antigenic uveitis, pulmonary cirrhosis, POEMS syndrome, polyarteritis nodosa, types I, II, and III; Primary chronic polyarthritis, polychondritis (e.g., refractory or relapsing polychondritis), polyendocrine autoimmune disease, polyendocrine insufficiency, polyglandular syndrome, optionally autoimmune polyglandular polyendocrinopathy syndrome (or polyendocrinopathy syndrome), polymyalgia rheumatica, polymyositis, polymyositis/dermatomyositis, polyneuropathy, acute polyradiculitis, postcardiotomy syndrome, posterior uveitis, or Autoimmune uveitis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, post-streptococcal nephritis, post-vaccination syndrome, presenile dementia, primary biliary cirrhosis, primary hypothyroidism, primary idiopathic myxedema, primary lymphocytosis (including monoclonal B-cell lymphocytosis, optionally including benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS) ), primary myxedema, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), primary sclerosing cholangitis, progesterone dermatitis, progressive systemic sclerosis, proliferative arthritis, psoriasis, e.g. , plaque psoriasis, psoriasis, psoriatic arthritis, alveolar proteinosis, pulmonary infiltrative eosinophilia, true red blood cell anemia or aplasia (PRCA), pure red cell aplasia, purulent or non-suppurative sinuses inflammation, pustular psoriasis and nail psoriasis, pyelitis, pyoderma gangrenosum, Kelvain thyroiditis, Raynaud phenomenon, reactive arthritis, recurrent miscarriage, reduced blood pressure response, reflex sympathetic dystrophy, refractory sprue, lighter disease or syndrome, relapsing polychondritis, reperfusion injury of myocardial tissue or other tissues, reperfusion injury, respiratory distress syndrome, restless legs syndrome, retinal autoimmunity, retroperitoneal fibrosis, Raynaud's syndrome, rheumatic disease, rheumatism Fever, rheumatoid arthritis, rheumatoid arthritis, rheumatoid spondylitis, rubella virus infection, Sumpter's syndrome, sarcoidosis, schistosomiasis, Schmidt syndrome, SCID, and Epstein-Barr virus related diseases, sclera, sclera scleroderma, sclerodactyl, scleroderma, optionally systemic scleroderma, sclerosing cholangitis, disseminated sclerosis, systemic sclerosis, sensorineural hearing loss, seronegative Spondyloarthritis, Sheehan syndrome, Schulman syndrome, silicosis, Sjögren's syndrome, sperm and testicular autoimmunity, sphenoid sinusitis, Stevens-Johnson syndrome, stiff man (or stiff person) syndrome, subacute bacterial endocarditis ( SBE), subacute cutaneous lupus erythematosus, sudden deafness, Suzak syndrome, Sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus (SLE) or systemic lupus erythematosus, cutaneous SLE, systemic Necrotizing vasculitis, ANCA-associated vasculitis, optionally Churg-Strauss vasculitis or syndrome (CSS), spinal fistula, Takayasu arteritis, telangiectasia, temporal arteritis/giant cell arteritis, obstructive thrombus Thrombocytopenias, including thrombotic thrombocytopenic purpura (TTP) and autoimmune or immune-mediated thrombocytopenias, such as idiopathic thrombocytopenic purpura (ITP) (chronic or acute (including ITP), thrombocytopenic purpura (TTP), thyrotoxicosis, tissue injury, Trosser-Hunt syndrome, toxic epidermal necrolysis, toxic shock syndrome, blood transfusion reaction, transient infantile hypogammaglobulinemia, transverse myelitis, traverse myelitis, tropical pulmonary eosinophilia, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), urticaria, optionally Chronic idiopathic urticaria, including chronic allergic urticaria and chronic autoimmune urticaria, uveitis, anterior uveitis, retinal uveitis, valvular inflammation, vascular dysfunction, vasculitis, spondyloarthritis, bullous Used in conjunction with therapeutic drugs, such as drugs used to treat skin diseases, vitiligo, Wegener's granulomatosis (GPA), Wiskott-Aldrich syndrome, or X-linked hyperIgM syndrome can be done.

The subject ADCs and novel corticosteroids of formula I, II, or III, and the corticosteroid linkers they contain, are useful for treating inflammation and inflammation, including, by way of example, autoimmune disorders, inflammatory disorders, infectious diseases, and cancer. It can be used for both prophylactic and/or therapeutic treatment of diseases associated with. A preferred use of the subject ADCs is for the treatment of chronic diseases associated with inflammation. Moreover, as shown in the Examples, in the specific case of VISTA antibodies containing ADCs, it is quite unexpected that these ADCs are contained therein (binding to VISTA-expressing cells in physiological conditions and at pH The short pK of anti-VISTA antibodies (not engineered to alter or optimize binding or increase half-life), i.e., typically about 2.3 days or less in cynomolgus monkeys, typically Despite a pK of only a few hours in human VISTA-engineered mice, it has been found to maintain potency (PD) for a much longer period of time compared to the much shorter half-life (pK) of antibodies.

As shown herein, VISTA ADC conjugates according to the invention when evaluated in in vitro and in vivo models provide a PD/PK ratio of at least 14:1 and even greater than or equal to 28:1 in immune cells. It has been proven that (In reality, the PD/PK ratio may be higher because the rodent or non-human primate was euthanized when PD was determined, thus not allowing a longer evaluation of efficacy.) ).

Although Applicants do not wish to be bound by any belief, VISTA ADCs containing the subject corticosteroid receptor agonist compounds of Formulas I, II, or III target VISTA, such as macrophages, T cells, and Tregs. It is theorized that very large amounts are delivered in VISTA-expressing cells, as well as other VISTA-expressing immune cells, including immune cells with long cell turnover (weeks, months, or more). Essentially, a depot effect is created within a specific type of immune cell, i.e., a large amount of the subject ADC or its "depot" due to the very high surface expression of VISTA, It is thought to be internalized by immune cells, such as macrophages and bone marrow cells. This in turn causes this depot containing internalized ADC to be slowly metabolized or cleaved within immune cells, apparently by cellular enzymes, for example. In vivo studies disclosed herein clearly demonstrate that metabolism or cleavage of internalized ADCs according to the present invention occurs for 1 week, 2 weeks, 4 weeks, or longer in rodents or primates, thereby , show that it can provide gradual and prolonged release of a therapeutically effective amount of steroid payload within the host's immune cells. This occurs despite the fact that no appreciable amount of ADC should remain in the serum by that time (i.e., based on the short pK of the ADC and the antibodies therein). , not enough ADC molecules will still remain in the peripheral circulation to induce a therapeutically significant effect on immunity).

Furthermore, although these observations are quite surprising, drug metabolism generally occurs much more rapidly in rodents than in primates (i.e., drug metabolism is much more rapid in humans than in rodents). Furthermore, because VISTA expression and the levels of VISTA-expressing immune cells are similar in rodents and human and non-human primates, the ADCs of interest are similar in humans and other primates. It is expected to have a PD/PK ratio of 20% or even higher. Accordingly, the subject ADCs should be well suited for therapeutic applications where long-term drug efficacy is desired or required.

Furthermore, Applicants have demonstrated that the ADCs according to the present invention compare favorably with previously reported ADCs due to the properties afforded by the novel glucocorticosteroid and steroid agonist linkers of formulas (I), (II), and (III). It possesses unique advantages, namely that the ADC containing it does not appear to be prone to aggregation, provides a high DAR, and furthermore internalizes immune cells very effectively, in which It has been found that it provides a large amount of active payload, resulting in rapid and long-term efficacy. Notwithstanding the foregoing, ADCs according to the invention with low DARs, e.g. 4.0 or less, are also shown herein to exhibit good efficacy; alternatively, e.g. It can be used for therapy because it has great development potential.

As mentioned above, another preferred use of the subject ADCs and novel glucocorticosteroids of Formula I, II, or III is for acute use, ie, to treat acute inflammation. As shown in the Examples, the glucocorticosteroids and ADCs of formula I, II or III according to the invention have a rapid onset of efficacy, for example they are as effective as within 2 hours after administration. Can induce rapid anti-inflammatory effects. Acute use of the subject anti-VISTA ADCs has demonstrated that the subject anti-VISTA ADCs effectively target and internalize neutrophils, rather than non-target cells, inducing anti-inflammatory effects. Therefore, it is even more advantageous. This is particularly beneficial in acute use, as neutrophils are involved in the early stages of the inflammatory response, and therefore the subject glucocorticosteroids and containing ADCs of formula I, II or III are also suitable for acute It is also well suited for treating inflammatory indications.

Another preferred use of the subject ADCs and novel corticosteroids of formula I, II or III is for maintenance therapy. In the specific case of VISTA ADC, VISTA can be used to target both activated and non-activated (naïve) immune cells, such as monocytes, macrophages, T cells, T regs, CD4 T cells, CD8 T cells, neutrophils. , expressed on eosinophils, dendritic cells, NK cells, and myeloid cells (by which VISTA is constitutively expressed), the subject ADC can be administered regularly over long periods of time. , such administration will induce anti-inflammatory activity both when the treated subject is in the active phase of the inflammatory response as well as when the subject is in disease remission. This is because many chronic autoimmune, allergic, and inflammatory disorders have periods of activity during which patients experience inflammation and other symptoms or pathological reactions associated with inflammation and the disease. or therapeutically beneficial because disease symptoms, including pathological reactions, are known to be absent or characterized by periods of much less severe remission (i.e., remission/relapsing or paroxysmal) It is. The ADC of interest may contain both activated and non-activated immune cells, such as monocytes, macrophages, T cells, T regs, CD4 T cells, CD8 T cells, neutrophils, eosinophils, dendritic cells. , NK cells, and bone marrow cells, it is expected that patients treated with the subject ADCs may maintain disease remission more effectively, i.e., the duration of remission should be more prolonged. and/or the active phase of the disease manifests in a much less severe form due to sustained anti-inflammatory efficacy of the subject ADCs on target immune cells both during active disease and during remission. obtain.

Furthermore, the subject ADC should be well suited for long-term or chronic use as there is no effect on non-target cells, ie, non-immune cells. As shown in the Examples below, the subject ADCs act virtually exclusively on target immune cells rather than non-immune cells (some anti-inflammatory activity was detected in the liver; (likely explained by the fact that it contains immune cells).

Also, in embodiments where the subject ADCs have short pKs (but surprisingly still have long PDs), the subject ADCs do not remain in the serum for long periods of time, i.e., they bind rapidly to immune cells. Clearly, ADC is efficiently and rapidly taken up by immune cells in large quantities and slowly metabolized within these immune cells, thereby delivering its anti-inflammatory payload and maintaining its potency over a long period of time. It is. Therefore, since the subject ADCs are present in the peripheral circulation for only a short period of time, the subject ADCs have long pKs because the antibodies they contain possess long PKs (which is conventional for most therapeutic antibodies). Compared to ADCs that have a limited number of opportunities to interact with non-target cells. A specific example is an ADC including Humira, which, like most conventional therapeutic antibodies, has a long pK, ie, about 1 month.

Still further, in some embodiments, the effectiveness of an ADC according to the invention (particularly an anti-VISTA ADC according to the invention comprising an FcR and an Fc region engineered to impair complement fixation) is determined by the anti-inflammatory payload. , for example, the subject ADCs should be well suited for long-term or chronic use. Essentially, antibodies in such cases, such as anti-VISTA antibodies, serve only a targeting function, ie, promote binding and internalization of the ADC by target immune cells. However, binding of such ADCs to VISTA-expressing immune cells does not modulate VISTA activity, i.e., anti-VISTA antibodies containing Fc engineered to eliminate Fc cross-linking do not agonize or antagonize VISTA activity. do not. [This is in contrast to existing ADCs for the delivery of steroids that contain antibodies that elicit a biological effect upon binding to a target antigen (such as the Humira ADC). ] This should be beneficial from a dosing point of view since the efficacy of the ADC depends solely on the anti-inflammatory payload. This also means that anti-VISTA or other immune cell antigen-targeting antibodies may, in some cases, otherwise be undesirable in the context of drugs aimed at mitigating inflammation, such as inflammatory cytokines. It is further therapeutically beneficial because it can induce a response.

Acute and chronic autoimmune and inflammatory indications for which the subject ADCs may be used are previously mentioned, among others Acquired Aplastic Anemia+, Acquired Hemophilia+, Acute Disseminated Encephalomyelitis (ADEM) +, acute hemorrhagic leukoencephalitis (AHLE)/Hurst's disease+, primary agammaglobulinemia+, alopecia areata+, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis+, antiphospholipid syndrome (APS) )+, arteriosclerosis, autism spectrum disorder (ASD), autoimmune Addison's disease (AAD)+, autoimmune autonomic neuropathy/autoimmune autonomic ganglionopathy (AAG), autoimmune encephalitis+, Autoimmune gastritis, autoimmune hemolytic anemia (AIHA)+, autohepatitis (AIH)+, autoimmune hyperlipidemia, autoimmune hypophysitis/lymphocytic hypophysitis+, autoimmune Inner ear disease (AIED)+, autoimmune lymphoproliferative syndrome (ALPS)+, autoimmune myocarditis, autoimmune oophoritis+, autoimmune orchitis+, autoimmune pancreatitis (AIP)/immunoglobulin G4 related disease (IgG4-RD)+, type I, type II, and type III autoimmune polyglandular syndrome+, autoimmune progesterone dermatitis+, autoimmune sudden deafness (SNHL), atonia, Addison's disease, Adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune Encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune Urticaria, axonal and neuronal neuropathy (AMAN), Barro's disease, Behcet's disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating Chronic Relapsing Polyneuritis (CIDP), Chronic Relapsing Multiple Osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial Pemphigoid, Cogan Syndrome, Cold Agglutinin Disease , congenital heart block, Coxsackie myocarditis, CREST syndrome, type 1 diabetes, dermatitis herpetiformis, dermatomyositis, Debic's disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, fibrosing alveolitis, Cellular myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or gestational pemphigoid (PG), hidraden abscess (HS) (opposite acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion Body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, flatus lichen, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (including nephritis and skin), chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease ( MCTD), Mooren's ulcer, Mucka-Habermann disease, multiple motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasia gravis, myelin oligodendrocyte glycoprotein antibody disorder, myositis, narcolepsy, neonatal lupus, optic nerve Myelitis, neutropenia, ocular cicatricial pemphigoid, optic neuritis, opsoclonus-myoclonus syndrome (OMS), relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal Hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Personage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, nodules Polyarteritis polyarteritis, polyglandular syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cholangitis, primary sclerosing bile duct inflammation, progesterone dermatitis, psoriasis, psoriatic arthritis, erythroblastic aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs. Syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial Endocarditis (SBE), Susac syndrome, sympathetic ophthalmitis (SO), Takayasu arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), thyroid eye disease (TED), Includes Tolosa Hunt syndrome (THS), transverse myelitis, type 1 diabetes, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

Preferred indications for which ADCs should be therapeutically effective include severe asthma, COPD, giant cell arteritis, ANKA vasculitis, and IBD (colitis (eg, ulcerative) and Crohn's disease). It is to be understood, of course, that the disease states identified herein are intended to be exemplary rather than exclusive.

The subject ADCs may be combined with other therapeutic agents, which may be administered in the same or different compositions, at the same time or at different times. For example, the subject ADCs can be administered in a therapeutic regimen that includes administration of a PD-1 or PD-L1 agonist, CTLA4-Ig, a cytokine, a cytokine agonist or antagonist, or another immunosuppressive receptor agonist or antagonist.

Other examples of specific immunosuppressive molecules that can be combined with ADCs according to the invention include antibodies that block co-stimulatory signals (e.g. against CD28 or ICOS), antibodies that activate inhibitory signals via CTLA4, and and/or antibodies to other immune cell markers (e.g., to CD40, CD40 ligand, or cytokines), fusion proteins (e.g., CTLA4-Fc or PD-1-Fc), and immunosuppressive drugs (e.g., rapamycin, cyclosporin A, or FK506).

Modified Fc Regions in ADCs According to the Invention As mentioned above, in some preferred embodiments of the invention, ADCs typically include antibodies that can be engineered to include modifications within the Fc region to alter one or more functional properties of the antibody. Additionally, in at least some embodiments of the invention, the ADC is chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or has its glycosylation altered and re-combined. , can be modified to alter one or more functional properties of the antibody. Such embodiments are described further below. The numbering of residues within the Fc region is that of Kabat's EU index.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, eg, increased or decreased. This approach is further described in US Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI can be varied, for example, to facilitate light and heavy chain assembly or to increase or decrease antibody stability.

In another embodiment, the Fc hinge region of the antibody is mutated to further reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are present at the CH2-CH3 domain interface of the Fc hinge fragment such that the antibody is impaired in staphylococcal protein A (SpA) binding compared to native Fc hinge domain SpA binding. introduced into the area. This approach is described in further detail in Ward et al., US Pat. No. 6,165,745.

In yet other embodiments, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may cause the antibody to have an altered affinity for the effector ligand, but not to the antigen of the parent antibody. Different amino acid residues can be substituted so as to retain binding ability. The effector ligand with modified affinity can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in US Pat. No. 5,624,821 and US Pat. No. 5,648,260 (both to Winter et al.).

In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 provide an antibody with altered C1q binding and/or reduced or reduced complement-dependent cytotoxicity (CDC). It may be substituted with a different amino acid residue to make it ineffective. This approach is described in further detail in US Pat. No. 6,194,551 by Idusogie et al.

In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered, thereby altering the ability of the antibody to fix complement. This approach is further described in PCT Publication No. WO 94/29351 by Bodmer et al.

In yet another example, the Fc region within an ADC is modified to increase the antibody's affinity for Fγ receptors by modifying one or more amino acids at the following positions: 238, 239, 248 , 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296 , 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376 , 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in PCT Publication No. WO 00/42072 by Presta. Furthermore, the binding sites of human IgG1 for FcyRI, FcyRII, FcyRIII, and FcRn have been mapped, and variants with improved binding have been described (Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcyRIII. In addition, the following combination variants have been shown to improve FcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A, and S298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E or M428L/N434S improve binding to FcRn and extend antibody circulating half-life (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316 (want to be).

In yet another embodiment, the antibodies within the ADC can be modified to abolish in vivo Fab arm exchange. Specifically, this process involves the exchange of IgG4 half molecules (one heavy chain and one light chain) between other IgG4 antibodies, effectively rendering the bispecific antibody functionally monovalent. bring to. Mutations to the hinge region and constant domain of the heavy chain can abolish this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105:9-19).

In yet another embodiment, the glycosylation of the antibody within the ADC is modified. For example, an aglycosylated antibody can be made (ie, the antibody lacks glycosylation). Glycosylation can be modified, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such non-glycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in further detail in Co et al., US Pat. No. 5,714,350 and US Pat. No. 6,350,861.

Additionally or alternatively, antibodies within the ADC can be made with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bipartite GlcNac structures. Such altered glycosylation patterns have been demonstrated to enhance the ADCC capabilities of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art, and include hosts that express recombinant antibodies according to at least some embodiments of the invention, thereby producing antibodies with altered glycosylation. Can be used as cells. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (a(1,6) fucosyltransferase), so that antibodies expressed in Ms704, Ms705, and Ms709 cell lines are Lacking fucose on top. Ms704, Ms705, and Ms709 FUT8 cell lines are generated by targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane- See Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene encoding fucosyltransferase, and antibodies expressed in such a cell line Demonstrates hypofucosylation by reducing or eliminating binding-associated enzymes. Hanai et al. also use cell lines with low or no enzymatic activity to add fucose to N-acetylglucosamine that binds to the Fc region of antibodies, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662). ). PCT Publication No. WO 03/035835 by Presta describes a mutant CHO cell line, Lecl3 cells, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed within its host cells. (See also Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication No. WO 99/54342 by Umana et al. shows that antibodies expressed in engineered cell lines exhibit an increase in bipartite GlcNac structures, which results in increased ADCC activity of the antibody. , P(l,4)-N-acetylglucosaminyltransferase III (GnTIII)) (see also Umana et al. (1999) Nat. Biotech. 17:176-180). Please refer). Alternatively, fucose residues of antibodies can be cleaved using fucosidase enzymes. For example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino, AL. et al. (1975) Biochem. 14:5516-23).

As mentioned in the exemplary embodiments, the Fc region of the antibody is mutated to impair FcR binding and, optionally, to impair complement fixation. These mutations include those contained in those exemplary antibodies. These mutations include L234A/L235A and L234A/L235A/E269R/K322A (IgG1 Fc), and V234A/G237A/P238s. Any or all of V309L/A330S/P331S (IgG2 Fc) are included.

Ex Vivo Use of ADCs According to the Invention According to at least some embodiments, immune cells, such as monocytes or myeloid cells, neutrophils, monocytes, T cells, B cells, among other immune cell types; NK cells, macrophages, mast cells, dendritic cells, Tregs, and other hematopoietic cells can be contacted with the subject's ADCs ex vivo to elicit an anti-inflammatory response, and the contacted cells are then transferred to the patient, For example, it can be injected into patients with allergic, autoimmune, or inflammatory conditions where reduction of inflammation is therapeutically desirable, modulating the immune response.

Exemplary Uses of the Subject ADCs and Pharmaceutical Compositions Containing for the Treatment of Autoimmune Diseases The ADCs and novel steroids of formula I, II, or III described herein can be used to treat immune system-related diseases. can be used. Optionally, the immune system related condition includes an autoimmune or inflammatory disease such as a previously identified disease, eg, transplant rejection, severe asthma, colitis or IBD, graft versus host disease. Optionally, the treatment is combined with another moiety useful for treating immune-related conditions.

Thus, treatment of multiple sclerosis using the subject ADCs may include, for example, any known therapeutic agent or method for treating multiple sclerosis, optionally as described herein. Can be combined.

Thus, treatment of rheumatoid arthritis or other arthritic conditions using the subject ADCs may include, for example, any known therapeutic agent or method for treating rheumatoid arthritis, as optionally described herein. can be combined with

Thus, treatment of IBD using the subject ADCs may be combined with any known therapeutic agent or method for treating IBD, eg, optionally as described herein.

Thus, treatment of psoriasis using the subject ADCs can be combined with any known therapeutic agent or method for treating psoriasis, eg, optionally as described herein.

Thus, treatment of type 1 diabetes using the subject ADCs may be combined, for example, with any known therapeutic agent or method for treating type 1 diabetes, optionally as described herein. obtain.

Thus, treatment of uveitis using the subject ADCs may be combined, for example, with any known therapeutic agent or method for treating uveitis, optionally as described herein. obtain.

Thus, treatment of psoriasis using the subject ADCs can be combined with any known therapeutic agent or method for treating psoriasis, eg, optionally as described herein.

Thus, treatment of Sjögren's syndrome using the subject ADCs may be combined with any known therapeutic agent or method for treating Sjögren's syndrome, for example, optionally as described herein.

Thus, treatment of systemic lupus erythematosus using the subject ADCs may be combined, for example, with any known therapeutic agent or method for treating systemic lupus erythematosus, optionally as described herein. obtain.

Thus, treatment of GVHD using the subject ADCs can be combined with any known therapeutic agent or method for treating GVHD, eg, optionally as described herein.

Accordingly, treatment of chronic or acute infections and/or hepatotoxicity associated therewith using the subject ADCs may include, for example, chronic or acute infections and/or hepatotoxicity, as optionally described herein. It may be combined with any known therapeutic agent or method for treating hepatotoxicity associated therewith.

Accordingly, treatment of chronic or acute severe asthma using the subject ADCs may include, for example, optionally in combination with any known therapeutic agent or method for treating severe asthma, as described herein. It can be done.

Accordingly, treatment of chronic or acute giant cell arteritis using the subject ADCs may include, for example, any known method for treating giant cell arteritis, as optionally described herein. may be combined with other therapeutic agents or methods.

Thus, treatment of chronic or acute ANKA vasculitis using the subject ADCs may include, for example, any known therapeutic agent or method for treating ANKA vasculitis, optionally as described herein. can be combined with

Accordingly, treatment of chronic or acute IBD (colitis and Crohn's disease) using the subject ADCs may include, for example, any known method for treating ANKA vasculitis, as optionally described herein. may be combined with other therapeutic agents or methods.

Again, the disease states identified herein, as well as the proposed treatments using the subject corticosteroid compounds, corticosteroid linker compounds, and ADCs containing the same, are exemplary and not exclusive. Please understand that this is my intention.

In the above therapy, preferably a subject with one of the aforementioned or other autoimmune or inflammatory conditions will be given an ADC according to the invention, thereby preventing or ameliorating the symptoms of the disease.

Pharmaceutical Compositions In another aspect, the present invention provides an ADC of formula I, II, or III according to the present invention or one of the novel steroid or corticosteroid linker compounds, or a combination thereof, optionally separately. compositions, e.g., pharmaceutical compositions, containing an immunosuppressant or other active agent. Accordingly, the invention features pharmaceutical compositions comprising a therapeutically effective amount of an ADC of Formula I, II, or III or a novel steroid or corticosteroid linker compound according to the invention. In particular, the invention provides pharmaceutical compositions comprising a therapeutically effective [anti-inflammatory] amount of one of the novel steroids of formula I, II, or III according to the invention or a corticosteroid linker compound containing or an ADC containing Characterize things.

The term "therapeutically effective amount" refers to an amount of an agent according to the invention that is effective to treat a disease or disorder in a mammal. The therapeutic agents of the invention can be provided to a subject alone or as part of a pharmaceutical composition in which they are mixed with a pharmaceutically acceptable carrier. In many cases, ADCs according to the invention will be used in combination with other immunotherapeutics or other therapeutic agents useful in treating a particular condition.

A composition is said to be "pharmaceutically acceptable" if its administration can be tolerated by the recipient patient. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and Equivalents may be mentioned. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).

Such compositions include sterile water, buffered saline (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, and optionally detergents and solubilizers (e.g., polysorbate). 20, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimersol, benzyl alcohol), and bulking substances (e.g., lactose, mannitol). Non-aqueous solvents or vehicles may also be used as detailed below.

Examples of suitable aqueous and non-aqueous carriers that may be employed in pharmaceutical compositions according to at least some embodiments of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, and the like), and the like. suitable mixtures of, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Active compounds, i.e. monoclonal or polyclonal antibodies and antibody fragments, and conjugates containing the same, which bind specifically to any one of the VISTA proteins or bispecific molecules, depending on the route of administration, and /or alternative scaffolds may be coated with substances to protect the compound from the effects of acids and other natural conditions that may deactivate the compound. Pharmaceutical compounds according to at least some embodiments of the invention may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxic effects (e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as aliphatic mono- and dicarboxylic acids, phenyl Included are those derived from non-toxic organic acids such as substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine. , and equivalents.

Pharmaceutical compositions according to at least some embodiments of the invention may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like. , (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like, and (3) citric acid. metal chelating agents such as acids, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by the sterilization procedures described above and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like in the compositions. Additionally, long-term absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. To the extent that any conventional vehicle or agent is incompatible with the active compound, its use in pharmaceutical compositions according to at least some embodiments of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. . Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is to produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof, in vacuo. Drying and freeze-drying (lyophilization).

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. . Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is to produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof, in vacuo. Drying and freeze-drying (lyophilization).

Compositions of the invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired result. Preferred routes of administration for therapeutic agents according to at least some embodiments of the invention include intravascular delivery (e.g., injection or infusion), intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral, Enteral, rectal, pulmonary (e.g., inhalation), nasal, topical (including transdermal, buccal, and sublingual), intravesical, intravitreal, intraperitoneal, vaginal, cerebral delivery (e.g., intraventricular, intracerebral). and convection-enhanced diffusion), CNS delivery (e.g., intrathecal, perimedullary, and intraspinal), or parenteral (including subcutaneous, intramuscular, intramedullary, intravenous, and intradermal), transmucosal (e.g. , sublingual administration), or administration via an implant, or other parenteral routes of administration, such as by injection or infusion, or other delivery routes and/or forms known in the art. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, including intravenous, intramuscular, intraarterial, intrathecal, and intracapsular administration. including, but not limited to, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, intrathecal, intraspinal, epidural and intrasternal injections and infusions. Not done. In specific embodiments, proteins, therapeutic agents, or pharmaceutical compositions according to at least some embodiments of the invention may be administered intraperitoneally or intravenously.

Alternatively, the ADC according to the invention may be administered via parenteral routes, such as topical, epidermal, or mucosal routes of administration, for example intranasally, orally, vaginally, rectally, sublingually, or topically. can be administered directly.

Therapeutic compositions containing ADCs according to the invention can be administered using medical devices known in the art. For example, in preferred embodiments, therapeutic compositions according to at least some embodiments of the present invention are disclosed in U.S. Patent No. 5,399,163; using a needle hypodermic injection device such as the devices disclosed in U.S. Patent No. 5,064,413, U.S. Pat. can be administered. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Pat. No. 4,486,194 discloses a therapeutic device for administering drugs through the skin; U.S. Pat. No. 4,447,233 discloses a drug infusion pump for delivering drugs at precise infusion rates; U.S. Pat. No. 4,447,224 discloses a variable flow implantable infusion device for continuous drug delivery, U.S. Pat. No. 4,439,196 discloses an osmotic drug delivery system with multichamber compartments, and U.S. Pat. No. 4,475,196 is disclosed, which discloses a pressure drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, ADCs may be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that therapeutic compounds according to at least some embodiments of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of making liposomes, see, for example, US Patent Nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes can contain one or more moieties that are selectively transported to specific cells or organs, thus improving targeted drug delivery (see, eg, V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, eg, Low et al., US Pat. No. 5,416,016), mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (PG Bloeman et al. (1995) FEBS Lett. 357: 140, M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180), surfactant protein A acceptance pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090). Also, K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123;J. J. Killion, and I. J. See also Fidler (1994) Immunomethods 4:273.

As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and Equivalents may be mentioned. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound, ie the ADC according to the invention, may be coated with a substance to protect the compound from the action of acids and other natural conditions that may deactivate the compound. Pharmaceutical compounds according to at least some embodiments of the invention may include one or more pharmaceutically acceptable salts.

"Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxic effects (e.g., Berge, S.M., et al. (1977) See also J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as aliphatic mono- and dicarboxylic acids, phenyl Included are those derived from non-toxic organic acids such as substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine. , and equivalents.

Pharmaceutical compositions according to at least some embodiments of the invention may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like. , (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like, and (3) citric acid. metal chelating agents such as acids, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and non-aqueous carriers that may be employed in pharmaceutical compositions according to at least some embodiments of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, and the like), and the like. suitable mixtures of, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by the sterilization procedures described above and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like in the compositions. Additionally, long-term absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. To the extent that any conventional vehicle or agent is incompatible with the active compound, its use in pharmaceutical compositions according to at least some embodiments of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. . Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is to produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof, in vacuo. Drying and freeze-drying (lyophilization).

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. . Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is to produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof, in vacuo. Drying and freeze-drying (lyophilization).

The amount of active ingredient, i.e., an ADC or novel steroidal compound of the invention, that can be combined with the carrier materials to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. . The amount of active ingredient, ie, the ADC or novel steroidal compound of the invention, that can be combined with the carrier materials to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount will include about 0.01 percent to about 99 percent active ingredient, such as about 0.1 percent to about 70 percent, most preferably, in combination with a pharmaceutically acceptable carrier. , for example, will range from about 1 percent to about 30 percent active ingredient.

Dosage regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Good too. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suitable as unit doses for the subject being treated, each unit containing the desired treatment together with the required pharmaceutical carrier. Contains a predetermined amount of active compound calculated to produce an effect. The specification of the dosage unit form according to at least some embodiments of the present invention depends on (a) the unique characteristics of the active compound and the particular therapeutic effect achieved, and (b) such The formulation of active compounds is determined by and directly dependent on the limitations inherent in the art.

For administration of the ADCs disclosed herein, in some embodiments, the dosage range is generally based on the effectiveness of conventional steroid compounds, such as dexamethasone or budesonide, when administered via conventional routes. administration of an amount of an ADC that delivers the same or a lower amount of an anti-inflammatory agent, such as a steroid of Formula I, II, or III, for therapeutic efficacy, as compared to that required for That is, steroids are administered in naked or unconjugated form to treat a specific condition. In exemplary embodiments, the dosage range generally includes a reduced amount of an anti-inflammatory agent, such as dexamethasone or budesonide, for therapeutic effectiveness than if the AI were administered via traditional routes. For example, including the administration of an amount of ADC that delivers 10-90% of the The present ADCs are effectively delivered by the desired target immune cells and have a lower propensity to reach non-target cells, thereby reducing the required effective dose of steroid and/or reducing the effects on non-target cells. As expected, steroids are administered in naked or unconjugated form to treat specific conditions.

The ADCs disclosed herein can be administered on multiple occasions. Intervals between single doses can be, for example, every 3 to 5 days, weekly, biweekly, every 2 to 3 weeks, etc. In some methods, the dosage is adjusted to achieve the desired level of plasma steroid concentration. Determining effective dosing regimens for treatment or prophylaxis using the subject ADCs can be difficult, since in some cases the therapeutic activity of the subject ADCs is completely dominated by the anti-inflammatory payload, and the antibodies therein are It should be relatively easy compared to other ADCs to induce biological or therapeutic effects. (Essentially, in some cases the antibody targets and induces only the internalization of the subject's ADC into specific immune cells and does not itself induce an effect on immunity).

Alternatively, the ADC may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage amount and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic use, relatively low doses may be administered at relatively infrequent intervals over an extended period of time. Some patients may continue to receive treatment for the rest of their lives. For therapeutic applications, relatively high doses may be required at relatively short intervals until disease progression is reduced or terminated, preferably until the patient shows partial or complete improvement in disease symptoms. be. The patient may then be placed on a prophylactic regimen. As mentioned above, the subject ADCs are preferred for such use because they remain in the peripheral circulation for very short periods of time, do not bind to non-immune cells, and do not clearly induce toxicity to non-target cells.

The actual dosage level of the active ingredients in the pharmaceutical compositions of the invention will be effective to achieve the desired therapeutic response for the particular patient, composition, and mode of administration without being toxic to the patient. Variations may be made to obtain certain amounts of active ingredient. The selected dosage level will be used in conjunction with the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, and the particular composition employed. various pharmacokinetics, including other drugs, compounds, and/or materials, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical field; It will depend on scientific factors.

Exemplary Embodiments The present invention provides antibodies or antigen-binding fragments (“A”) that specifically bind to immune cell antigens, such as human V domain Ig inhibitor of T cell activation (human VISTA), cleavable and/or or a non-cleavable linker (“L”) and at least one small molecule anti-inflammatory agent (“AI”), optionally linking the linker to an anti-VISTA antibody or antibody fragment and at least one small molecule anti-inflammatory agent (“AI”); “AI”) (typically a steroid), which is a chemical moiety optionally used to connect a “heterobifunctional” or “heterotrifunctional” group; An antibody drug conjugate (ADC) is provided, the ADC having the formula:
represented by "A-(Q-L-AI) _n " or "(AI-L-Q) _n -A",
where "n" is at least 1 and can be as high as 12 or more, and the ADC or composition containing it, when administered to a subject in need thereof, expresses the antigen bound by the antibody. cells, such as VISTA-expressing immune cells, optionally monocytes, myeloid cells, T cells, Tregs, eosinophils, CD4 or CD8 T cells, neutrophils, optionally delivered to physiological Preferably, the antibody or antibody fragment, e.g. In vivo serum half-life in serum at physiological pH (approximately pH 7.5), optionally up to 70 hours in rodents (e.g., human VISTA knock-in mice or rats), up to 60 hours at physiological pH (approximately pH 7.5); Hours or less, 50 hours or less, 40 hours or less, 30 hours or less, 24 hours or less, 22 to 24 hours or less, 20 to 22 hours or less, 18 to 20 hours or less, 16 to 18 hours or less, 14 to 16 hours or less, 12 ~14 hours or less, 10-12 hours or less, 8-10 hours or less, 6-8 hours or less, 4-6 hours or less, 2-4 hours or less, 1-2 hours or less, 0.5-1.0 hours or less or an in vivo serum half-life in serum of 0.1 to 0.5 hours or less, and/or 3, 2.5, or 2.3 ± 0.7 days in primates (e.g., humans or cynomolgus monkeys). have

Exemplary cleavable and non-cleavable linkers that can be incorporated into the subject ADCs have been previously identified herein and are well known in the art. Specific types of linkers that can be used in ADCs according to the invention and examples of such types are further identified below.

As previously mentioned, steroid compounds are highly effective in inhibiting inflammation associated with different conditions such as autoimmunity, allergies, and inflammatory disorders, cancer, and infectious diseases, but in chronic treatment of diseases. Their usefulness is limited due to severe side effects that would be alleviated when incorporated into ADCs according to the present invention.

In particular, the invention includes ADCs according to the invention, where the AI includes steroids (glucocorticoid agonists) that include the following general structure:

Ｘが、フェニル、スピロ［３．３］ヘプタン、３～６員複素環、シクロアルキル、スピロアルキル、スピロヘテロシクロアルキル、二環式アルキル、ヘテロ二環式アルキル、［１．１．１］ビシクロペンタン、ビシクロ［２．２．２］オクタン、アダマンタン、及びキュバンから選択され、これらの各々を、独立して、Ｆ、Ｃｌ、Ｂｒ、Ｉ、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換することができ、その環構造の各々が、Ｎ、Ｓ、及びＯから選択される少なくとも１つの骨格ヘテロ原子を含み得、任意選択的に、１～４個のＣ_１～３アルキルまたはＣ１～３ペルフルオロアルキルで更に置換され、
Ｚが、フェニル、スピロ［３．３］ヘプタン、３～６員複素環、シクロアルキル、スピロアルキル、スピロヘテロシクロアルキル、二環式アルキル、ヘテロ二環式アルキル、［１．１．１］ビシクロペンタン、ビシクロ［２．２．２］オクタン、アダマンタン、及びキュバンから選択され、これらの各々を、独立して、Ｆ、Ｃｌ、Ｂｒ、Ｉ、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換することができ、その環構造の各々が、Ｎ、Ｓ、及びＯから選択される少なくとも１つの骨格ヘテロ原子を含み得、任意選択的に、１～４個のＣ_１～３アルキルまたはＣ１～３ペルフルオロアルキルで更に置換され、
Ｙが、ＣＨＲ_１、Ｏ、Ｓ、及びＮＲ_１から選択され、
Ｅが、ＣＨ_２及びＯから選択され、
Ｇが、ＣＨ及びＮから選択され、
更に、ＧがＣＨであり、Ｘがフェニルであるときに、Ｚがフェニルではなく、
ＸへのＧの結合が、任意選択的にＣ_１～３アルキル及びエチレンオキシドから選択され得、これらの各々が、独立して、Ｎ、Ｓ、及びＯから選択される１～４個のヘテロ原子で置換され得、任意選択的に、１～４個のＣ_１～３アルキルで更に置換され、
ＺへのＸの結合が、Ｘ及びＺ上の任意の利用可能な位置を占有し得、
置換基ＮＲ_１Ｒ_２が、Ｚ上の任意の利用可能な位置を占有し得、
Ｒ_１が、Ｈ、１～８個の炭素の直鎖または分岐アルキル、アリール、及びヘテロアリール基から選択され、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ_２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｒ_１がＨであるとき、Ｒ_２が、Ｈ、１～８個の炭素の直鎖または分岐アルキル、アリール、及びヘテロアリール基から選択され得、アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ_２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｒ_１がＨ、１～８個の炭素の直鎖もしくは分岐アルキル、またはヘテロアリールであるとき、Ｒ_２が、
［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］_ｍ－［Ｃ＝Ｏ］－［Ｖ］_ｋ－Ｊ、
Ｃ＝Ｏ）ＯＣＨ_２－ｐ－アミノフェニル－Ｎ－Ｖ－Ｊ、
（Ｃ＝Ｏ）ＯＣＨ_２－ｐ－アミノフェニル－Ｎ－［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］_ｍ－［Ｃ＝Ｏ］－［Ｖ］_ｋ－Ｊ、及び
［Ｖ］_ｋ－（Ｃ＝Ｏ）ＯＣＨ_２－ｐ－アミノフェニル－Ｎ－［（Ｃ＝Ｏ）ＣＨ（Ｗ）ＮＨ］_ｍ－［Ｃ＝Ｏ］－Ｊ、
式中、ｍ＝１～６であり、ｋ＝０～１であり、Ｗの各順列が、独立して、Ｈ、ｎ＝１～４である［（ＣＨ_２）_ｎＲ_３］、Ｒ_３で終端する分岐アルキル鎖、及び１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基から選択され得、
Ｒ_３が、Ｈ、メチル、エチル、イソプロピル、ＯＨ、Ｏ－アルキル、ＮＨ_２、ＮＨ－アルキル、Ｎ－ジアルキル、ＳＨ、Ｓ－アルキル、グアニジン、尿素、カルボン酸、カルボキサミド、カルボン酸エステル、置換または非置換アリール、置換または非置換ヘテロアリールから選択され、当該アリール及びヘテロアリール置換基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－Ｏ－アルキル、－ＮＨ２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－から選択され得、
Ｖが、１～８個の炭素のアルキル鎖、１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基、１～８個の炭素を含む直鎖または分岐アルキル基、－Ｏ－アルキル、カルボン酸、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－、１～３アミノ酸配列であって、各アミノ酸が、独立して、Ｇｌｕ、Ｇｌｙ、Ａｓｎ、Ａｓｐ、Ｇｌｎ、Ｌｅｕ、Ｌｙｓ、Ａｌａ、βＡｌａ、Ｐｈｅ、Ｖａｌ、及びＣｉｔから選択される、１～３アミノ酸配列、アリール、ならびにヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－ＮＨ_２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｊが、－ＮＨ_２、Ｎ_３、チオ、シクロオクチン、－ＯＨ、－ＣＯ_２Ｈ、トランスシクロオクテン、アルキニル、プロパルギル、

から選択される反応基であり、
式中、Ｒ_３２が、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ_３３が、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ_３４が、Ｈ、Ｍｅ、テトラジン－Ｈ、及びテトラジン－Ｍｅであり、
Ｒ_５が、－ＣＨ_２ＯＨ、－ＣＨ_２ＳＨ、－ＣＨ_２Ｃｌ、－ＳＣＨ_２Ｃｌ、－ＳＣＨ_２Ｆ、－ＳＣＨ_２ＣＦ_３、ヒドロキシ、－ＯＣＨ_２ＣＮ、－ＯＣＨ_２Ｃｌ、－ＯＣＨ_２Ｆ、－ＯＣＨ_３、－ＯＣＨ_２ＣＨ_３、－ＳＣＨ_２ＣＮ、及び

からなる群から選択され、
Ｒ_６及びＲ_７が、独立して、水素及びＣ_１～１０アルキルから選択され、
Ｑが、Ｈ、

Ｒ_８が１～８個の炭素の直鎖もしくは分岐アルキルである、Ｃ（Ｏ）Ｒ_８、またはｎ＝１～４であり、Ｒ_４＝Ｈ、アルキルもしくは分岐アルキル、またはＰ（Ｏ）ＯＲ_４である、（Ｃ＝Ｏ）ＮＲ_４ＣＨ_ｎＮＲ_４（Ｃ＝Ｏ）ＯＣＨ_２－（Ｖ）_ｎ－Ｊであり得、
Ａ_１及びＡ_２が、独立して、Ｈ及びＦから選択され、別途明記しない限り、全ての可能な立体異性体が特許請求され、更に任意選択的に、Ｘ及びＺが、独立して、フェニル、スピロ［３．３］ヘプタン、［１．１．１］ビシクロペンタン、及びビシクロ［２．２．２］オクタンから選択され、Ｙが、ＣＨ_２及びＯから選択され、Ｗの順列が、独立して、ＣＨ_２ＣＨ_２ＣＯ_２Ｈ及びＨから選択され、更に、ＧがＣＨであり、Ｘがフェニルであるときに、Ｚがフェニルではない、グルココルチコイドアゴニスト化合物、
または
式（ＩＩ）の構造を保有するグルココルチコイドアゴニスト化合物であって、

式中、
Ｙが、ＣＨ_２及びＯから選択され、
Ｅが、ＣＨ_２及びＯから選択され、
Ｇが、ＣＨ及びＮから選択され、
Ｌが、Ｈ及びＦから選択され、
Ｒ_５が、－ＣＨ_２ＯＨ、－ＳＣＨ_２Ｆ、及び

から選択され、
Ａ_１及びＡ_２が、独立して、Ｈ及びＦから選択され、
Ｖが、１～８個の炭素のアルキル鎖、１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基、１～８個の炭素を含む直鎖または分岐アルキル基、－Ｏ－アルキル、カルボン酸、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－、１～３アミノ酸配列であって、各アミノ酸が、独立して、Ｇｌｕ、Ｇｌｙ、Ａｓｎ、Ａｓｐ、Ｇｌｎ、Ｌｅｕ、Ｌｙｓ、Ａｌａ、βＡｌａ、Ｐｈｅ、Ｖａｌ、及びＣｉｔから選択される、１～３アミノ酸配列、アリール、ならびにヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－ＮＨ_２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｊが、－ＮＨ_２、Ｎ_３、チオ、シクロオクチン、－ＯＨ、－ＣＯ_２Ｈ、トランスシクロオクテン、アルキニル、プロパルギル、

から選択される反応基であり、
式中、Ｒ_３２が、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ_３３が、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ_３４が、Ｈ、Ｍｅ、テトラジン－Ｈ、及びテトラジン－Ｍｅである、グルココルチコイドアゴニスト化合物、
または
式（ＩＩＩ）の構造を保有するグルココルチコイドアゴニスト化合物であって、

式中、
Ｙが、ＣＨ_２及びＯから選択され、
Ｅが、ＣＨ_２及びＯから選択され、
Ｇが、ＣＨ及びＮから選択され、
Ｌが、Ｈ及びＦから選択され、
Ｒ_５が、－ＣＨ_２ＯＨ、－ＳＣＨ_２Ｆ、及び

から選択され、
Ａ_１及びＡ_２が、独立して、Ｈ及びＦから選択され、
Ｖが、１～８個の炭素のアルキル鎖、１～１３個の単位を含む直鎖または分岐ポリエチレンオキシド基、１～８個の炭素を含む直鎖または分岐アルキル基、－Ｏ－アルキル、カルボン酸、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－、１～３アミノ酸配列であって、各アミノ酸が、独立して、Ｇｌｕ、Ｇｌｙ、Ａｓｎ、Ａｓｐ、Ｇｌｎ、Ｌｅｕ、Ｌｙｓ、Ａｌａ、βＡｌａ、Ｐｈｅ、Ｖａｌ、及びＣｉｔから選択される、１～３アミノ酸配列、アリール、ならびにヘテロアリール基から選択され得、当該アリール及びヘテロアリール基が、アルキル、ハロアルキル、ハロゲン、ビフェニル、ニトロ、ニトリル、－ＯＨ、－ＮＨ_２、アルキルアミノ、ジアルキルアミノ、チオール、チオアルキル、グアニジン、尿素、カルボン酸、アルコキシル、カルボキサミド、カルボン酸エステル、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、ジアルキルアミノＣ（Ｏ）－から選択される官能基で置換され得、
Ｊが、－ＮＨ_２、Ｎ_３、チオ、シクロオクチン、－ＯＨ、－ＣＯ_２Ｈ、トランスシクロオクテン、アルキニル、プロパルギル、

から選択される反応基であり、
式中、Ｒ_３２が、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ_３３が、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ_３４が、Ｈ、Ｍｅ、テトラジン－Ｈ、及びテトラジン－Ｍｅである、グルココルチコイドアゴニスト化合物が提供される。 Specifically, herein, a glucocorticoid agonist compound having the following structure of formula (I),

X is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C ₁ further substituted with _~3 alkyl or C1-3 perfluoroalkyl,
Z is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C ₁ further substituted with _~3 alkyl or C1-3 perfluoroalkyl,
Y is selected from CHR ₁ , O, S, and NR ₁ ,
E is selected from CH ₂ and O;
G is selected from CH and N;
Furthermore, when G is CH and X is phenyl, Z is not phenyl,
The bond _of G to optionally further substituted with 1 to 4 C _1-3 alkyl,
the bond of X to Z may occupy any available position on X and Z;
The substituent NR ₁ R ₂ may occupy any available position on Z;
R ₁ is selected from H, linear or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and the aryl and heteroaryl groups are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, - OH, -O-alkyl, _-NH2 , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C( O)-, and dialkylamino C(O)-,
When R ₁ is H, R ₂ may be selected from H, straight chain or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and the aryl and heteroaryl groups are alkyl, haloalkyl, halogen , biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH ₂ , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O) may be substituted with a functional group selected from O-, alkylamino-C(O)-, and dialkylamino-C(O)-;
When R ₁ is H, linear or branched alkyl of 1 to 8 carbons, or heteroaryl, R ₂ is
[(C=O)CH(W)NH] _m -[C=O]-[V] _k -J,
C=O)OCH ₂ -p-aminophenyl-NVJ,
(C=O)OCH ₂ -p-aminophenyl-N-[(C=O)CH(W)NH] _m -[C=O]-[V] _k -J, and [V] _k -(C =O)OCH ₂ -p-aminophenyl-N-[(C=O)CH(W)NH] _m -[C=O]-J,
where m=1 to 6, k=0 to 1, and each permutation of W is independently H, n=1 to 4 [(CH ₂ ) _n R ₃ ], R ₃ branched alkyl chains terminating in and linear or branched polyethylene oxide groups containing from 1 to 13 units;
R ₃ is H, methyl, ethyl, isopropyl, OH, O-alkyl, NH ₂ , NH-alkyl, N-dialkyl, SH, S-alkyl, guanidine, urea, carboxylic acid, carboxamide, carboxylic acid ester, substituted or selected from unsubstituted aryl, substituted or unsubstituted heteroaryl, wherein the aryl and heteroaryl substituents are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH2, alkylamino, dialkyl selected from amino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino-C(O)- Gain,
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein the aryl and The heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH ₂ , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl may be substituted with a functional group selected from -C(O)O-, alkylamino-C(O)-, dialkylamino C(O)-,
J is -NH ₂ , N ₃ , thio, cyclooctyne, -OH, -CO ₂ H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R ₃₂ is selected from Cl, Br, F, mesylate, and tosylate, and R ₃₃ is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-( 4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, R ₃₄ is H, Me, tetrazine-H, and tetrazine-Me;
R ₅ is -CH ₂ OH, -CH ₂ SH, -CH ₂ Cl, -SCH ₂ Cl, -SCH ₂ F, -SCH ₂ CF ₃ , hydroxy, -OCH ₂ CN, -OCH ₂ Cl, -OCH ₂ F, -OCH ₃ , -OCH ₂ CH ₃ , -SCH ₂ CN, and

selected from the group consisting of
R ₆ and R ₇ are independently selected from hydrogen and C _1-10 alkyl;
Q is H,

R ₈ is straight chain or branched alkyl of 1 to 8 carbons, C(O)R ₈ , or n=1 to 4, and R ₄ =H, alkyl or branched alkyl, or P(O)OR ₄ , (C=O)NR ₄ CH _n NR ₄ (C=O)OCH ₂ -(V) _n -J,
A ₁ and A ₂ are independently selected from H and F; unless otherwise specified, all possible stereoisomers are claimed, and further optionally, X and Z are independently selected from H and F; phenyl, spiro[3.3]heptane, [1.1.1]bicyclopentane, and bicyclo[2.2.2]octane, Y is selected from CH ₂ and O, and the permutations of W are glucocorticoid agonist compounds independently selected from CH ₂ CH ₂ CO ₂ H and H; further, when G is CH and X is phenyl, Z is not phenyl;
or a glucocorticoid agonist compound having the structure of formula (II),

During the ceremony,
Y is selected from CH ₂ and O;
E is selected from CH ₂ and O;
G is selected from CH and N;
L is selected from H and F;
R ₅ is -CH ₂ OH, -SCH ₂ F, and

selected from
A ₁ and A ₂ are independently selected from H and F;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein the aryl and The heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH ₂ , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl may be substituted with a functional group selected from -C(O)O-, alkylamino-C(O)-, dialkylamino C(O)-,
J is -NH ₂ , N ₃ , thio, cyclooctyne, -OH, -CO ₂ H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R ₃₂ is selected from Cl, Br, F, mesylate, and tosylate, and R ₃₃ is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-( glucocorticoid agonist compounds selected from (4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R ₃₄ is H, Me, tetrazine-H, and tetrazine-Me;
or a glucocorticoid agonist compound having the structure of formula (III),

During the ceremony,
Y is selected from CH ₂ and O;
E is selected from CH ₂ and O;
G is selected from CH and N;
L is selected from H and F;
R ₅ is -CH ₂ OH, -SCH ₂ F, and

selected from
A ₁ and A ₂ are independently selected from H and F;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein the aryl and The heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH ₂ , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl may be substituted with a functional group selected from -C(O)O-, alkylamino-C(O)-, dialkylamino C(O)-,
J is -NH ₂ , N ₃ , thio, cyclooctyne, -OH, -CO ₂ H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R ₃₂ is selected from Cl, Br, F, mesylate, and tosylate, and R ₃₃ is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-( 4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R ₃₄ is H, Me, tetrazine-H, and tetrazine-Me. Ru.

式中、
Ａｂ＝抗体、好ましくは、ヒト免疫細胞に結合する抗体、好ましくは、生理学的ｐＨにおいてヒトＶＩＳＴＡ免疫細胞に結合する抗ＶＩＳＴＡ抗体、
Ｌ＝リンカー、
ＡＡ＝単一、二重、または三重アミノ酸配列、
ＮＨペイロード＝

Ｒ^ＥＧが、独立して、水素、アルキル、ビフェニル、－ＣＦ_３、－ＮＯ_２、－ＣＮ、フルオロ、ブロモ、クロロ、アルコキシル、アルキルアミノ、ジアルキルアミノ、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－からなる群から選択され、

Ａｂ＝抗体、
Ｌ＝リンカー、
ＡＡ＝単一、二重、または三重アミノ酸配列、
ＮＨペイロード＝

Ｒ^ＥＧが、独立して、水素、アルキル、ビフェニル、－ＣＦ_３、－ＮＯ_２、－ＣＮ、フルオロ、ブロモ、クロロ、アルコキシル、アルキルアミノ、ジアルキルアミノ、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－からなる群から選択され、

Ａｂ＝抗体、
Ｌ＝リンカー、
ＡＡ＝単一、二重、もしくは三重アミノ酸配列、または存在しない、
ＮＨペイロード＝

Ｒ^ＥＧが、独立して、水素、アルキル、ビフェニル、－ＣＦ_３、－ＮＯ_２、－ＣＮ、フルオロ、ブロモ、クロロ、アルコキシル、アルキルアミノ、ジアルキルアミノ、アルキル－Ｃ（Ｏ）Ｏ－、アルキルアミノ－Ｃ（Ｏ）－、及びジアルキルアミノＣ（Ｏ）－からなる群から選択され、
ＲＴ＝ＡＡまたは

Ａｂ＝抗体、任意選択的に、抗ヒトＶＩＳＴＡ抗体、
Ｌ＝リンカー、
ＡＡ＝単一、二重、または三重アミノ酸配列、
Ｏペイロード＝

Ａｂ＝抗体、
Ｌ＝リンカー、
ＡＡ＝単一、二重、もしくは三重アミノ酸配列、または存在しない、
Ｏペイロード＝

ＲＴ＝ＡＡまたは

である。
Ｂ．アミノ酸（ＡＡ）リンカー
（Ｉ）カテプシンによって切断された配列
ａ．単一アミノ酸リンカー

ｂ．ジペプチドリンカー

ｃ．トリペプチドリンカー

（Ｉ）レグマイン切断可能リンカー

式中、
Ｌ＝リンカー
Ａｂ＝抗体

は、ペイロードまたは犠牲リンカーへの付着点を示す。 I. Exemplary Linkers As mentioned above, linkers can be incorporated into different ADCs according to the invention. Such linkers have been previously identified in the definition section where "linker" is defined. Additionally, exemplary linkers that can be incorporated into ADCs according to the invention are identified below.
A. sacrificial linker ADC

During the ceremony,
Ab = antibody, preferably an antibody that binds to human immune cells, preferably an anti-VISTA antibody that binds to human VISTA immune cells at physiological pH;
L = linker,
AA = single, double, or triple amino acid sequence;
NH payload =

R ^EG is independently hydrogen, alkyl, biphenyl, -CF ₃ , -NO ₂ , -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino -C(O)-, and dialkylamino C(O)-,

Ab=antibody,
L = linker,
AA = single, double, or triple amino acid sequence;
NH payload=

R ^EG is independently hydrogen, alkyl, biphenyl, -CF ₃ , -NO ₂ , -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino -C(O)-, and dialkylamino C(O)-,

Ab=antibody,
L = linker,
AA = single, double or triple amino acid sequence or absent;
NH payload=

R ^EG is independently hydrogen, alkyl, biphenyl, -CF ₃ , -NO ₂ , -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino -C(O)-, and dialkylamino C(O)-,
RT=AA or

Ab = antibody, optionally anti-human VISTA antibody,
L = linker,
AA = single, double, or triple amino acid sequence;
O payload =

Ab=antibody,
L = linker,
AA = single, double or triple amino acid sequence or absent;
O payload=

RT=AA or

It is.
B. Sequence cleaved by amino acid (AA) linker (I) cathepsin a. single amino acid linker

b. dipeptide linker

c. tripeptide linker

(I) Legumain cleavable linker

During the ceremony,
L = linker Ab = antibody

indicates the point of attachment to the payload or victim linker.

リンカー＝Ｑ、Ｒ_１またはＲ_２
Ｊは、Ａｂ上の相補性官能基と反応して抗体薬物コンジュゲートを形成するために好適な官能基である。
Ｊは、

から選択され、

は、Ｑ、Ｒ_１またはＲ_２から選択されるリンカーへのＪの付着点を示す。
式中、Ｒ_３２は、Ｃｌ、Ｂｒ、Ｆ、メシル酸塩、及びトシル酸塩から選択され、Ｒ_３３は、Ｃｌ、Ｂｒ、Ｉ、Ｆ、ＯＨ、－Ｏ－Ｎ－スクシンイミジル、－Ｏ－（４－ニトロフェニル）、－Ｏ－ペンタフルオロフェニル、または－Ｏ－テトラフルオロフェニルから選択され、Ｒ_３４は、Ｈ、Ｍｅ、またはピリジルから選択され、
－ＯＨ基を、例えば、アスパラギン酸またはグルタミン酸側鎖上の抗体上のカルボキシ基でエステル化することができ、
－ＣＯ２Ｈ基を、－ＯＨ基でエステル化するか、または抗体上の（例えば、リジン側鎖上の）アミノ基でアミド化することができ、
Ｎ－ヒドロキシスクシンイミド基は、機能的に活性化カルボキシル基であり、（例えば、リジンからの）アミノ基との反応によって便宜的にアミド化することができ、
マレイミド基を、マイケル付加反応において、（例えば、システインから、またはスルフヒドリル官能性を導入するための抗体の化学修飾から）抗体上の－ＳＨ基とコンジュゲートすることができる。 II. Exemplary Antibody Conjugation Strategies Different conjugation strategies can be used to conjugate anti-VISTA antibodies to linkers and payloads (steroids or other anti-inflammatory compounds). Detailed synthetic methods for producing exemplary ADC and linker payloads are provided in the Examples. Additionally, exemplary conjugation strategies are provided below.
(I) Payload-Linker-J
The payload is:

Linker = Q, R ₁ or R ₂
J is a functional group suitable for reacting with a complementary functional group on the Ab to form an antibody drug conjugate.
J is

selected from

indicates the point of attachment of J to a linker selected from Q, R ₁ or R ₂ .
where R ₃₂ is selected from Cl, Br, F, mesylate, and tosylate, and R ₃₃ is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-( 4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, R ₃₄ is selected from H, Me, or pyridyl;
The -OH group can be esterified, for example, with a carboxy group on the antibody on the aspartic acid or glutamic acid side chain;
The -CO2H group can be esterified with an -OH group or amidated with an amino group on the antibody (e.g. on a lysine side chain);
The N-hydroxysuccinimide group is a functionally activated carboxyl group and can be conveniently amidated by reaction with an amino group (e.g. from lysine);
A maleimide group can be conjugated with a -SH group on an antibody (eg, from cysteine or from chemical modification of the antibody to introduce sulfhydryl functionality) in a Michael addition reaction.

If the antibody does not have a cysteine -SH available for conjugation, the ε-amino group in the side chain of the lysine residue can be substituted with 2-iminothiolane or N-succinimidyl-3-(2-pyridyldithio). A free thiol (-SH) group can be introduced by reaction with propionate ("SPDP") to create a cysteine surrogate. Thiol groups can be reacted with maleimide or other nucleophilic acceptor groups to achieve conjugation.

The antibody Ab is modified with 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (“SMCC”) or its sulfonated variant sulfo-SMCC, both available from Sigma-Aldrich, A maleimide group can be introduced there. Conjugation can then be accomplished using a drug linker compound with a --SH group on the linker.

Copper-free "click chemistry" in which an azide group (-N3) is added across a strained cyclooctyne to form a 1,2,3-triazole ring. The azide can be placed on the antibody and cyclooctyne on the drug linker moiety, or vice versa. A preferred cyclooctyne group is dibenzocyclooctyne (DBCO).

An unnatural amino acid is introduced into the antibody, and the unnatural amino acid provides the functionality for conjugation with a reactive functional group on the drug moiety. For example, the unnatural amino acid p-acetylphenylalanine can be incorporated into antibodies or other polypeptides. The ketone group within p-acetylphenylalanine can be the site of conjugation via oxime formation with the hydroxylamino group on the linker drug moiety. Alternatively, the unnatural amino acid p-azidophenylalanine (or p-azidomethyl-l-phenylalanine) is incorporated into the antibody to provide an azido functionality for conjugation via click chemistry with DBCO, 1,2, A 3-triazole ring can be formed.

Another example is the incorporation of unnatural amino acids containing strained alkenes norbornene, trans cyclooctene, or cyclopropene, which undergo reverse electron demand Diels-Alder "click chemistry" reaction with tetrazine to The formula diazine product can be formed.

Another conjugation technique uses the enzyme transglutaminase (preferably bacterial transglutaminase or BTG from Streptomyces mobaraensis). BTG forms an amide bond between the side chain carboxamide of glutamine (amine acceptor) and an alkylene amino group (amine donor), which can be, for example, the epsilon-amino group or 5-amino-n-pentyl group of lysine. . In a typical conjugation reaction, a glutamine residue is located on the antibody, while an alkylene amino group is located on the linker drug moiety.

は、抗体またはその抗原結合断片への付着点を示す。

は、システイン残基の硫黄原子、またはその薬学的に許容される塩、互変異性体、立体異性体、及び／または立体異性体の混合物を介した、抗体またはその抗原結合断片への付着点を示す。

は、リンカーまたはＡＡへの付着点を示す。 III. Exemplary Antibody Conjugates Antibodies or fragments targeting desired immune cell antigens, such as anti-VISTA antibodies (optional binding to human VISTA at physiological pH and having a previously defined short PK); The present invention optionally comprises one or more cleavable and/or non-cleavable linkers and one or more payloads (steroids or other anti-inflammatory compounds) optionally attached to the sacrificial linker. ADC conjugates can be produced using the detailed synthetic methods described above and disclosed in the Examples. Some exemplary ADC structures and conjugation methods are provided below.
(I) Preferred embodiment

indicates the point of attachment to the antibody or antigen-binding fragment thereof.

is the point of attachment to the antibody or antigen-binding fragment thereof via the sulfur atom of a cysteine residue, or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof. shows.

indicates the point of attachment to the linker or AA.

IV. Exemplary Payload Linker Structures Different payloads (steroids or other anti-inflammatory compounds) attached to the linker can be produced using the detailed synthetic methods described above and disclosed in the Examples. Exemplary payload linker structures are provided below as well as in FIGS. 118A-O.

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）
Ｊ＝ブロモアセチル

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）
Ｊ＝ジベンジルシクロオクチン

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）
Ｊ＝ヒドロキシスクシンイミド

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）
Ｊ＝マレイミド

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）
Ｊ＝テトラジン

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）
Ｊ＝ＴＧコンジュゲーション

(I) Payload-Linker-J
During the ceremony,
Linker = Protease cleavable sequence (AA)
J = alkoxyamine

During the ceremony,
Linker = Protease cleavable sequence (AA)
J=bromoacetyl

During the ceremony,
Linker = Protease cleavable sequence (AA)
J = dibenzylcyclooctyne

During the ceremony,
Linker = Protease cleavable sequence (AA)
J = hydroxysuccinimide

During the ceremony,
Linker = Protease cleavable sequence (AA)
J=maleimide

During the ceremony,
Linker = Protease cleavable sequence (AA)
J=tetrazine

During the ceremony,
Linker = Protease cleavable sequence (AA)
J=TG conjugation

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝ブロモアセチル

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝ジベンジルシクロオクチン

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝ヒドロキシスクシンイミド

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝マレイミド

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝テトラジン

式中、
リンカー＝プロテアーゼ切断可能配列（ＡＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝アミン

(II) Payload-Linker-J
During the ceremony,
Linker = protease cleavable sequence (AA) + sacrificial linker para-aminobenzyl (PAB)
J = alkoxyamine

During the ceremony,
Linker = protease cleavable sequence (AA) + sacrificial linker para-aminobenzyl (PAB)
J=bromoacetyl

During the ceremony,
Linker = protease cleavable sequence (AA) + sacrificial linker para-aminobenzyl (PAB)
J = dibenzylcyclooctyne

During the ceremony,
Linker = protease cleavable sequence (AA) + sacrificial linker para-aminobenzyl (PAB)
J = hydroxysuccinimide

During the ceremony,
Linker = protease cleavable sequence (AA) + sacrificial linker para-aminobenzyl (PAB)
J=maleimide

During the ceremony,
Linker = protease cleavable sequence (AA) + sacrificial linker para-aminobenzyl (PAB)
J=tetrazine

During the ceremony,
Linker = protease cleavable sequence (AA) + sacrificial linker para-aminobenzyl (PAB)
J = amine

式中、
リンカー＝グルクロニダーゼ切断可能糖（ＧｌｃＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝ブロモアセチル

式中、
リンカー＝グルクロニダーゼ切断可能糖（ＧｌｃＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝ジベンジルシクロオクチン

式中、
リンカー＝グルクロニダーゼ切断可能糖（ＧｌｃＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝ヒドロキシスクシンイミド

式中、
リンカー＝グルクロニダーゼ切断可能糖（ＧｌｃＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝マレイミド

式中、
リンカー＝グルクロニダーゼ切断可能糖（ＧｌｃＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝テトラジン

式中、
リンカー＝グルクロニダーゼ切断可能糖（ＧｌｃＡ）＋犠牲リンカーパラアミノベンジル（ＰＡＢ）
Ｊ＝アミン

(II) Payload-Linker-J
During the ceremony,
Linker = glucuronidase cleavable sugar (GlcA) + sacrificial linker para-aminobenzyl (PAB)
J = alkoxyamine

During the ceremony,
Linker = glucuronidase cleavable sugar (GlcA) + sacrificial linker para-aminobenzyl (PAB)
J=bromoacetyl

During the ceremony,
Linker = glucuronidase cleavable sugar (GlcA) + sacrificial linker para-aminobenzyl (PAB)
J = dibenzylcyclooctyne

During the ceremony,
Linker = glucuronidase cleavable sugar (GlcA) + sacrificial linker para-aminobenzyl (PAB)
J = hydroxysuccinimide

During the ceremony,
Linker = glucuronidase cleavable sugar (GlcA) + sacrificial linker para-aminobenzyl (PAB)
J=maleimide

During the ceremony,
Linker = glucuronidase cleavable sugar (GlcA) + sacrificial linker para-aminobenzyl (PAB)
J=tetrazine

During the ceremony,
Linker = glucuronidase cleavable sugar (GlcA) + sacrificial linker para-aminobenzyl (PAB)
J = amine

ブロモアセチル

マレイミド

ジベンゾシクロオクチン

テトラジン

アミン

(IV) Payload-Linker-J
Alternative site of linker-J to payload (C11-OH).
Alkoxyamine INX-SM-3 is used as an example payload

Bromoacetyl

maleimide

dibenzocyclooctyne

Tetrazine

amine

ブロモアセチル

マレイミド

ジベンゾシクロオクチン

テトラジン

アミン

(IV) Payload-Linker-J
Alternative site of linker-J to payload (C17).
Alkoxyamine INX-SM-3 is used as an example payload

Bromoacetyl

maleimide

dibenzocyclooctyne

Tetrazine

amine

アジド＋ジベンゾシクロオクチンコンジュゲーション（Ｃ１１－ＯＨ結合）

ハロアセチル＋システインコンジュゲーション（Ｃ１１－ＯＨ結合）

マレイミド＋システインコンジュゲーション（Ｃ１１－ＯＨ結合）

テトラジン＋トランスシクロオクテンコンジュゲーション（Ｃ１１－ＯＨ結合）

トランスグルタミナーゼを使用したアミン＋グルタミンコンジュゲーション（Ｃ１１－ＯＨ結合）

アルコキシアミン＋ケトンコンジュゲーション（Ｃ１７）

アジド＋ジベンゾシクロオクチンコンジュゲーション（Ｃ１７）

システインへのハロアセチルコンジュゲーション（Ｃ１７）

システインへのマレイミドコンジュゲーション（Ｃ１７）

テトラジン＋トランスシクロオクテン（Ｃ１７）

トランスグルタミナーゼを使用したアミン＋グルタミンコンジュゲーション（Ｃ１７）

Ｎ結合ペイロードリンカー－Ａｂ ＡＤＣ

V. Exemplary payload linker-Ab conjugate (INX-SM-3 is an exemplary payload)
an antibody or antibody fragment that binds an antigen expressed by an immune cell, optionally an anti-VISTA antibody or fragment having pH binding/PK properties as described herein, one or more linkers, and one or more Different ADC conjugates containing a payload (steroid or other anti-inflammatory compound) can be produced using the detailed synthetic methods described above and disclosed in the Examples. Several exemplary ADCs are provided below, including an exemplary steroid payload (INX-SM-3). Other exemplary ADCs are disclosed in the Examples and may further include other steroid payloads according to the invention, ie, those containing steroid compounds of Formula I, II, or III as disclosed herein.
Alkoxyamine + ketone conjugation (C11-OH bond)

Azide + dibenzocyclooctyne conjugation (C11-OH bond)

Haloacetyl + cysteine conjugation (C11-OH bond)

Maleimide + cysteine conjugation (C11-OH bond)

Tetrazine + trans cyclooctene conjugation (C11-OH bond)

Amine+glutamine conjugation (C11-OH bond) using transglutaminase

Alkoxyamine + ketone conjugation (C17)

Azide + dibenzocyclooctyne conjugation (C17)

Haloacetyl conjugation to cysteine (C17)

Maleimide conjugation to cysteine (C17)

Tetrazine + trans cyclooctene (C17)

Amine+glutamine conjugation using transglutaminase (C17)

N-linked payload linker-Ab ADC

Steroid Payloads of Formulas I, II, and III, Steroid Linker Payloads, and Therapeutic Uses of ADCs Containing ADCs containing synthetic glucocorticoid agonists according to the present invention may be produced as described in the Examples above and below. In some exemplary embodiments, the antibody contained therein comprises an anti-human VISTA antibody or fragment that binds to immune cells at physiological pH and also possesses a short PK, or that is associated with another immune system. An antibody or antibody fragment that binds to a cellular antigen, preferably an antibody or antibody fragment that efficiently internalizes target immune cells and preferably does not substantially bind to or internalize non-target cells. obtain. In general, the subject ADCs contain a steroid of Formula I, II, or III or a steroid linker containing same and an antigen expressed on a specific type of cell, preferably expressed only on a target immune cell. or at a substantially higher level, e.g., at least 2-fold, 4-fold, 10-fold higher, or more, on target cells compared to non-target cells expressing the antigen. will include antibodies or antibody fragments that bind.

These ADCs may be used to treat inflammation, allergy, autoimmunity, and, by way of example, the autoimmune disorders, inflammatory disorders, allergic disorders, and cancer conditions disclosed herein. It may be used for prophylactic and/or therapeutic treatment of related diseases. Again, a preferred use of the subject ADCs, including those containing steroids of Formula I, II, or III, is for the treatment of chronic diseases associated with inflammation or autoimmunity.

As provided herein, the subject VISTA targeting ADCs binds to VISTA-expressing cells in physiological conditions and has not been engineered to alter or optimize pH binding. Short pKs of anti-VISTA antibodies, i.e., typically less than about 2.3 days in cynomolgus monkeys, less than 70 hours, less than 60 hours, less than 50 hours, less than 40 hours, less than 30 hours, and less than 24 hours in human VISTA-engineered mice. 22 to 24 hours or less, 20 to 22 hours or less, 18 to 20 hours or less, 16 to 18 hours or less, 14 to 16 hours or less, 12 to 14 hours or less, 10 to 12 hours or less, 8 to 10 hours or less, Despite a pK of 6 to 8 hours or less, 4 to 6 hours or less, 2 to 4 hours or less, 1 to 2 hours or less, 0.5 to 1.0 hours or less, or 0.1 to 0.5 hours or less , have been found to maintain potency (PD) for a much longer period of time compared to the much shorter half-life (PK) of antibodies.

As shown herein, ADC conjugates of the invention comprising such anti-VISTA antibodies when evaluated in an in vivo model provide a PD/PK ratio of at least 14:1 and 28:1. It has been proven that Again, although Applicants do not wish to be bound to the belief, the subject ADCs comprising such anti-VISTA antibodies or antibody fragments may have long cell turnover (weeks, months, or even longer). It is theorized that VISTA is delivered in very large quantities in targeted VISTA-expressing cells such as macrophages. Essentially, a depot effect may be created, i.e., a large amount of the subject ADC is internalized into VISTA-expressing immune cells, i.e., due to the very high surface expression of VISTA, so that the ADC, e.g. It is thought to be slowly metabolized or cleaved by enzymes, resulting in gradual and prolonged release of therapeutically effective amounts of steroid payload within immune cells. However, ADCs according to the invention, i.e. containing a steroid payload of formula I, II or III, may include antibodies or antibody fragments that bind to other antigens, typically antigens expressed on human immune cells. The emphasis should be on gaining. Indeed, in the Examples, Applicants show that ADCs containing steroid payloads according to the invention, including antibodies targeting different immune cell antigens, also possess desirable efficacy properties.

Having described the invention, the following examples are provided to further illustrate the invention and its inherent advantages.

The following examples describe exemplary embodiments of the invention.

リンカーＡの合成のためのスキーム

Example 1: Synthesis and Characterization of Exemplary Steroid-Anti-VISTA Antibody ConjugatesA. synthesis

Scheme for the synthesis of linker A

ジクロロメタン／アセトニトリル（５００ｍＬ／１００ｍＬ）中の化合物１（３．０ｇ、７．６４ｍｍｏｌ、１．０当量）の溶液に、環状無水物（３．０ｇ、３０．５８ｍｍｏｌ、４．０当量）及びＤＭＡＰ（１．８ｇ、１５．２９ｍｍｏｌ、２．０当量）を添加した。反応混合物を室温で２時間撹拌させ、混合物を減圧下で濃縮した。残渣を、ＤＣＭ／ＭｅＯＨ（１０％～１５％）＋０．１％ＡｃＯＨで溶出されたシリカゲル上のカラムクロマトグラフィーによって精製し、白色の固体として化合物２（３．２ｇ、８５％）を得た。
ＴＬＣ：ＤＣＭ／ＭｅＯＨ＝１０：１、ＵＶ
Ｒ_ｆ（化合物１）＝０．４５
Ｒ_ｆ（化合物２）＝０．３０
ＬＣ－ＭＳ：３９４．４０（Ｍ＋１）

Procedure General procedure for the preparation of compound 2

To a solution of compound 1 (3.0 g, 7.64 mmol, 1.0 eq.) in dichloromethane/acetonitrile (500 mL/100 mL) was added the cyclic anhydride (3.0 g, 30.58 mmol, 4.0 eq.) and DMAP ( 1.8g, 15.29mmol, 2.0eq) was added. The reaction mixture was allowed to stir at room temperature for 2 hours and the mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (10%-15%) + 0.1% AcOH to give compound 2 (3.2 g, 85%) as a white solid.
TLC:DCM/MeOH=10:1, UV
R _f (compound 1) = 0.45
R _f (compound 2) = 0.30
LC-MS: 394.40 (M+1)

To a solution of 2 (220 mg, 0.45 mmol) and 3 (230 mg, 0.67 mmol) in NMP (4 mL) was added HATU (342 mg, 0.90 mmol) and DIPEA (232 mg, 1.8 mmol). The mixture was stirred at room temperature for 5 hours. The mixture was purified by preparative HPLC (ACN/H2O, 0.1% HCOOH) to yield Linker A (122 mg, 39%).
LCMS:703[M+H],
¹ H NMR (CDCl ₃ , 300 MHz) (δ, ppm) 7.20 (d, J = 9.0 Hz, 1 H), 6.73 (s, 2 H), 6.52 (br, 1 H) , 6.33 (d, J = 9.0 Hz, 1 H), 6.11 (s, 1 H), 4.91 (q, J = 17.3 Hz, 2 H), 4.35 (d = 9.3 Hz, 1 H), 3.76 - 3.42 (m, 10 H), 3.03 (m, 1 H), 2.79 (m, 2 H), 2.65 - 2. 56 (m, 3 H), 2.42 - 2.06 (m, 7 H), 1.84 - 1.63 (m, 3 H), 1.22 (m, 1 H), 1.02 (s , 3 H), 0.90 (d, J = 7.2 Hz, 3 H).
^19F NMR ( _CDCl3 ) (δ, ppm) -166.09 (q).

General scheme for the preparation of conjugates with linker A. To conjugate 8 linkers A per antibody, the antibodies were buffer exchanged into PBS buffer pH 7.4 at a concentration of 10 mg/mL, followed by 7. An equivalent amount of TCEP was added and incubated at 37°C for 2 hours. The reduced antibody was then buffer exchanged by a PD-10 column (GE Healthcare) with 50 mM borate buffer at pH 8.0 containing 2 mM EDTA, followed by 12 equivalents of Linker A (in DMSO). (freshly prepared as a 10 mM stock solution) was added and the reaction was left in a tube revolver at 10 rpm for 1 hour at ambient temperature. The conjugate containing 8 linkers A per antibody was purified using a PD-10 desalting column with PBS buffer, pH 7.4. After elution, the conjugate was further buffer exchanged and concentrated to the desired concentration using an Amicon Ultra 15 mL centrifugal filter with a 30 kDa molecular weight cutoff (MWCO). Mass spectrometry To determine the drug-to-antibody ratio (DAR), the conjugate was incubated with 25mM DTT for 30 minutes at 37°C. The reduced conjugate was diluted 50 times in water and analyzed on a Waters ACQUITY UPLC interfaced to a Xevo G2-S QToF mass spectrometer. Unconvolved masses were obtained using Waters MassLynx 4.2 Software. The drug-to-antibody ratio (DAR) was calculated using the weighted average of the peak intensities corresponding to each drug-loaded species using the following formula:
DAR=Σ(drug loading distribution (%) for each Ab with drug loading n)(n)/100

SEC Method Size exclusion high performance liquid chromatography using a 20 minute isocratic method with a mobile phase of 0.2 M sodium phosphate, 0.2 M potassium chloride, 15 w/v isopropanol at pH 6.8. The purity of the conjugate was determined by (SEC-HPLC). A 10 μL injection volume was loaded onto a TSKgel SuperSW3000 column at a constant flow rate of 0.35 mL/min. The chromatographs were integrated based on elution time and the purity of the monomeric conjugate species was calculated.

After synthesis of the antibody drug conjugates (ADCs) described above, the naked antibodies and ADCs underwent a quality control process to evaluate and confirm conjugation, ability to bind to VISTA, and endotoxin levels. We also synthesized a control pH-dependent binding anti-VISTA antibody (767-IgG1.3 antibody) that possesses a relatively long in vivo half-life in physiological conditions and analyzed it using peptide mapping to determine its sequence identity and The pH-dependent binding was confirmed.

B. Confirmation of drug-antibody ratio and purity by SEC Conjugation levels, presence of high molecular weight (HMW) aggregates, and endotoxin levels were evaluated for conjugates after conjugation with Linker A (assay performed by Abzena). Briefly, conjugation levels were assessed via reverse phase HPLC, mass spectrometry, or both. The level of HMW aggregates was assessed via a size exclusion column. Endotoxin levels were assessed via the Charles River endosafe-PTS system using LAL test cartridges.

200 μg of control anti-human VISTA antibody (767-IgG1.3) was incubated with trypsin (1/20 trypsin/protein) for 14 hours at 23°C or with Lys-C (1/50 Lys-C/protein). Digestion was performed for either 14 hours at 37°C. 80 μg of sample was analyzed by mass spectrometry on an Agilent QTOF 6530B. Sequence searches were performed using BioConfirm 9.0.

C. ELISA results 1. ELISA for determination of pH-specific binding
96-well flat bottom plates (Thermo Scientific Nunc Immuno Maxisorp, Cat. No. 442404) were coated with 767-IgG1.3 or INX200 diluted to 1 μg/mL in PBS for 1 hour at room temperature (RT). Wells were washed three times with PT (PBS with 0.05% Tween 20) and then blocked with PTB (PBS with 0.05% Tween 20 and 1% BSA) for 1.5 hours at room temperature.

Biotinylated hIX50 (human VISTA ECD produced by Aragen Bioscience, biotinylated in ImmuNext) was added to citric acid/containing 0.05% Tween 20 and 1% BSA at pH 6.1, 6.7 or 7.5. Diluted in phosphate salt (CPTB) to a range of 1000-0.001 ng/mL. Wells were washed three times with citric acid/phosphate (CPT) containing 0.05% Tween 20 at pH 6.1, 6.7 or 7.5, then biotinylated hIX50 was added to the wells and incubated at room temperature. and incubated for 1 hour.

After washing three times with CPT at pH 6.1, 6.7 or 7.5, streptavidin conjugated to HRP (Southern Biotech, catalog number 7100-05) was added to the solution at pH 6.1, 6.7 or 7.5. was used as a detection reagent at a dilution of 1/2000 in CPTB and incubated for 1 hour at room temperature. After three washes with CPT at pH 6.1, 6.7 or 7.5, the ELISA reaction was revealed using TMB (Thermo Scientific, Cat. No. 34028) as a colorimetric substrate. After 5 minutes at room temperature, the reaction was stopped with 1M H2S04.

2. ELISA for confirmation of VISTA binding of naked and drug-conjugated antibodies
A 96-well flat bottom plate (same as above) was coated with hIX50 (human VISTA ECD, produced by Aragen Bioscience for ImmuNext) at 1 μg/ml in PBS for 1 hour at room temperature (RT). After washing three times, wells were blocked with PTB for 1 hour at room temperature.

INX200, INX200A, INX201, INX201A, 767-IgG1.3 or 767-IgG1.3A was diluted in PTB to a range of 500-0.03, 100-0.02, or 400 or 0.1 ng/mL. Wells were washed three times with PT, then diluted antibodies were added to the wells and incubated for 1 hour at room temperature.

After washing three times with PT, mouse anti-human Kappa-HRP (Southern Biotech, catalog number 9230-05) was used at a 1/2000 dilution in PTB as the detection reagent and incubated for 1 hour at room temperature. After three washes, ELISA reactions were revealed using TMB substrate. After 5 minutes _at room temperature, the reaction was stopped with 1M _H2SO4 .

D. Conjugation and SEC Purity Levels of Antibodies Evaluated Conjugation of Linker A involves complete reduction of interchain disulfides followed by complete modification by Linker A (confirmed by mass spectrometry [MS] conjugation evaluation). ). Minimal HMW aggregates were detected as assessed by size exclusion chromatography (SEC) and reported as % purity (see Table 1 below).

E. Peptide mapping of 767-IgG1.3 As shown in Figure 1, tryptic digestion resulted in 85.6% light chain sequence coverage and 76.1% heavy chain sequence coverage. The figure shows the sequence of 767-IgG1.3 with (A) light chain (85.6% coverage) and (B) heavy chain (76.1% coverage) with identified tryptic peptides underlined. .

As shown in Figure 2, Lys-C digestion resulted in 63.3% light chain sequence coverage and 76.3% heavy chain sequence coverage. The figure shows the sequence of 767-IgG1.3 with (A) light chain (63.3% coverage) and (B) heavy chain (76.3% coverage) with identified tryptic peptides underlined. .

The total combined sequence coverage between the trypsin and Lys-C digestion strategies was 91.7% light chain sequence coverage and 80.8% heavy chain sequence coverage. Both the light and heavy chains match the intended sequence as described in WO2018/169993A1. Based on this, it was confirmed that the cloned and expressed sequence was that of 767-IgG1.3.

F. Comparison of VISTA binding of anti-VISTA antibodies at different pH conditions As shown in Figure 3, plate-bound 767-IgG1.3 and INX200 were confirmed to possess opposite anti-VISTA pH-dependent binding properties. Specifically, Figure 3 shows that at pH 7.5 (physiological pH) 767-IgG1.3 has minimal binding to soluble VISTA, and binding to VISTA is clearly higher at pH 6.7. The highest degree of binding was at pH 6.1, indicating that the highest degree of binding was at pH 6.1 (lowest pH tested. In contrast, INX200 had the highest degree of binding to soluble VISTA at physiological pH; (again, relative binding at pH 6.7 and pH 6.1 was compared). Therefore, 767-IgG1.3 and INX200 have opposite pH-dependent binding properties. show.

G. Effect of drug conjugation on VISTA binding The anti-VISTA antibody drug conjugates identified above underwent complete reduction of interchain disulfides by approximately DAR 8 conjugation to dexamethasone-based linker A in in vitro and in vivo ADC studies. This has been proven. Furthermore, as shown in Figures 4A-C, DAR 8 conjugation with linker A resulted in negligible binding of INX200A, INX201A, or 767-IgG1.3A to VISTA compared to naked antibody. It was shown that this effect was significant (Fig. 4A to C).

H. Conclusion The experiments and data described above confirm that the control 767-IgG1.3 antibody contains the same sequence and functional properties (pH-dependent binding) as the previously described 767-IgG1.3 antibody. These data demonstrate that all anti-VISTA antibody drug conjugates made underwent complete cysteine reduction and that DAR 8 conjugation using dexamethasone-based linker A resulted in minimal HMW aggregate formation (as assessed by SEC purity). We further showed that such conjugation had negligible effect on the binding of the antibody drug conjugate to human VISTA.

Example 2: In Vivo Characterization of Exemplary Anti-VISTA Drug Conjugates A. ConA Model Again, VISTA was chosen as a potential target for anti-inflammatory antibody drug conjugates (ADCs) because it is highly expressed on most hematopoietic cells, especially on bone marrow cells. The efficacy of the Dex-antibody drug conjugate was evaluated in order to evaluate its potential efficacy in the development of ADCs for potential use in treating autoimmune and inflammatory diseases. A short-term model of liver inflammation (ConA-induced hepatitis) was evaluated.

This model involves intravenous (i.v.) injection of the plant lectin concanavalin A (ConA) in mice and includes a widely used model for acute immune-mediated hepatitis in mice. In contrast to some other models of acute liver injury, ConA-induced damage is primarily driven by T cell activation and recruitment to the liver. Thus, the ConA model has unique features with respect to its pathogenesis and important similarities to immune-mediated hepatitis in humans, such as autoimmune hepatitis, acute viral hepatitis, or distinctly different entities of drug toxicity leading to immune activation. have sex. The ConA model is characterized by a burst of inflammatory cytokines that can be monitored as early as 6 hours after injection. By 24 hours, high levels of inflammatory cytokines can still be detected and liver damage/necrosis can be observed by histopathology. This model was exploited primarily by monitoring cytokine responses 6 hours after ConA injection. As discussed below and shown in the figures, these studies show that Dex treatment has a dose-dependent effect on G-CSF, IFNγ, IL-2, IL-6, IL-12p40, IL-12p70, and KC. Our study focused on measuring some of these cytokines, as they have been shown to have sexual effects.

B. Study Design In these experiments, mice received antibody or Dex treatment approximately 15 hours before disease onset. Concanavalin A administration was adjusted to produce acute but non-lethal inflammation at 6 hours and was established in preliminary experiments. Blood was collected 6 hours after intravenous injection of ConA and plasma was isolated for cytokine analysis.

The purpose of the in vivo study was to evaluate the relative efficacy of these INX human VISTA antibodies conjugated to dexamethasone via an esterase-sensitive linker compared to free Dex in ConA-induced hepatitis. In particular, in vivo studies have shown that anti-human VISTA antibodies, either naked or conjugated to dexamethasone (INX210 [silent IgG2 Fc], INX200 [silent IgG1 Fc], and 767.3- IgG1.3 [control pH-sensitive antibody]) (Experiments 1, 2, and 3, respectively).

These experiments were performed in human VISTA knock-in (hVISTA KI) mice. hVISTA KI mice have a knock-in human VISTA cDNA in place of the mouse VISTA gene and express human VISTA at both RNA and protein levels. Additionally, these experiments were performed in female and male mice to exclude the nullity of gender differences. All animals received treatment (antibody or dexamethasone) 15 hours before concanavalin A (ConA) injection. Mice were then bled 6 hours after ConA injection and cytokine responses were assessed as a marker of disease progression.

C. Methods and Materials Anti-VISTA Antibodies and Conjugates INX200: Possesses a very short serum half-life at physiological pH (see Table 7 below) and contains variable heavy and light sequences and an IgG1 Fc region included in Figure 8; A humanized anti-human VISTA antibody on a human IgG1/kappa backbone with L234A/L235A silencing mutations in the Fc region).

INX200A: INX200 conjugated to the dexamethasone drug via an interchain disulfide with a drug/antibody ratio (DAR) of approximately 8. The linker/payload (A) consists of an esterase-sensitive linker with a dexamethasone payload (described in Graverson et al, 2012).

INX201: has variable heavy and light sequences and an IgG1 Fc region contained in Figure 8, possesses a very short serum half-life at physiological pH (see Table 7 below), L234A/L235A/E269R/ in the Fc region. Humanized anti-human VISTA antibody on human IgG1/kappa backbone with K322A silencing mutation).

INX201A: INX201 antibody conjugated to the dexamethasone drug via an interchain disulfide with a drug/antibody ratio (DAR) of 8. The linker/payload (A) again consists of an esterase-sensitive linker with a dexamethasone payload (as described in Graverson et al, 2012).

INX210: possesses a very short serum half-life (see Table 7 below), has variable heavy and light sequences and an IgG1 Fc region included in Figure 8), V234A/G237A/P238S/H268A/V309L in the Fc region Humanized anti-human VISTA antibody on human IgG2/kappa backbone with /A330S/P331S silencing mutations (Vafa et al, 2014).

INX210A: INX210 antibody conjugated to a drug via an interchain disulfide with a drug/antibody ratio (DAR) of approximately 8. The linker/payload (A) again consists of an esterase-sensitive linker with a dexamethasone payload (as described in Graverson et al, 2012).

767-IgG1: with variable heavy and light sequences and an IgG1 Fc region included in Figure 8, with a much longer serum half-life (>24 hours in rodents and primates), L234A/ Control humanized anti-human VISTA antibody developed by Five Prime Therapeutics and Bristol-Myers Squibb Company on human IgG1/kappa with L235E/G237A silencing mutations. This antibody was designed to bind VISTA at low pH (eg pH 6), but to have minimal binding at physiological pH (pH 7.4) (WO2018/169993A1).

767-IgG1A: 767-IgG1 antibody conjugated to a drug via an interchain disulfide with a drug/antibody ratio (DAR) of approximately 8. The linker/payload (A) again consists of an esterase-sensitive linker with a dexamethasone payload (as described in Graverson et al, 2012).

Antibody administration:
All antibodies were diluted in PBS and injected intraperitoneally (i.p.) in a volume of 0.2 ml to deliver a dose of 10 mg/Kg.

Dexamethasone Dexamethasone (sterile injection from Phoenix, NDC 57319-519-05) was diluted in PBS and administered via intraperitoneal injection at 5, 2, 0.2 and 0.02 mg/Kg.

Concanavalin A
Concanavalin A was obtained from Sigma Aldrich (C2010). Depending on its lot, ConA can be more or less toxic, so preliminary experiments were always performed to define the best ConA administration to produce acute but non-lethal inflammation in 6 hours. It was 15 mg/Kg (lot number SLBX7517) in experiments 1 and 2, and 7.5 mg/Kg (lot number SLCC2664) in experiments 3 and 4.

Mice hVISTA mice were bred at Sage Labs (Boyertown, PA). Mice, 8-12 weeks old, were first transported in a quarantine facility for 3 weeks and then transferred to regular facilities. They were allowed to acclimate for 1-2 weeks before starting the experiment.

Blood Sampling and Preparation Peripheral blood was collected from the retroorbital cavity using a glass Pasteur pipette that was first rinsed with heparin to prevent clotting. Blood was then centrifuged at 400 rcf for 5 minutes and plasma was collected and stored at -80°C prior to cytokine analysis.

Plasma Cytokine Analysis Cytokine analysis was performed on 25 μl of plasma using the Millipore mouse 7plex platform.

Experiments 1 and 2: Cytokines included in the analysis for in vivo studies were G-CSF, IL-2 IFNγ, IL-6, IL-12p40, IL-12p70, and KC.

Experiment 3: For in vivo experiment 3, G-CSF (DY414-05, expected <100,000 pg/mL G-CSF, likely <50,000 pg/mL - kit detection level: 2000 pg/mL to 31.3 pg /mL) and KC (DY453-05, expected <120,000 pg/mL, likely <50,000 pg/mL - kit detection level: 1000 pg/mL to 15.6 pg/mL) using R&D Duo set ELISA Only G-CSF and KC were analyzed via .

D. Results Experiment 1: Efficacy of INX210A in ConA-induced hepatitis in female hVISTA KI mice Figure 5 shows G-CSF changes 6 hours after ConA in the peripheral blood of female hVISTA KI mice. Plasma concentrations (SEM; n = 5/group) measured using mouse 7plex (administration: Dex-0.2 = 0.2 mg/Kg, Dex-2 = 2 mg/Kg, INX210 at 10 mg/Kg and INX210A, [INX210A provided a dex payload of 0.2 mg/kg]).

As shown in the figure, treatment with INX210A had some (but not significant) efficacy in controlling ConA-induced G-CSF upregulation, comparable to Dex treatment at 5 mg/Kg. Indicated. In contrast, non-Dex-conjugated antibody INX210 or Dex administered at 0.2 mg/Kg (which is the molar equivalent of Dex delivered by INX210A) had no anti-inflammatory effect.

Data from six other cytokines were not included as they were too diverse for interpretation, as we observed a high level of within-group variability in ConA responses. This is not unexpected as the effects of ConA are highly dependent on the hormonal status of the animal when experiments are performed in female mice. Female mice may show higher susceptibility to ConA, but also show more variation in disease outcome. All subsequent ConA experiments were performed in male mice.

Experiment 2: Efficacy of INX210A in ConA-induced hepatitis in male hVISTA KI mice Figure 6 shows cytokine changes 6 hours after ConA in the peripheral blood of male hVISTA KI mice. Plasma concentrations measured using mouse 7plex (SEM; n=10/group, normal one-way ANOVA compared to ConA only group) (Dosing: Dex at 0.2 or 5 mg/Kg, 10 mg/Kg INX210 and INX210A).

As previously reported in the literature, male mice showed a more consistent cytokine response to ConA. Six of the seven cytokines analyzed showed a significant reduction (1-3 fold) compared to the untreated ConA group 6 hours after INX210A treatment (Figure 6). The reduction was intermediate between 0.2 mg/kg Dex (molar equivalent of INX210A dex payload) and 5 mg/kg Dex. In contrast, no efficacy was observed in the INX210 treated group.

Experiment 3: Dose response of INX200A on Concanavalin A-induced hepatitis in DDE1 male mice Figure 7 shows cytokine changes 6 hours after ConA in the peripheral blood of DDE1 male mice. In the experiment, cytokine plasma concentrations were measured using an ELISA assay (SD; n=6/group; one-way ANOVA compared to ConA only group) (dosing: 0.02, 0.2, or 2 mg/group). Dex in Kg, INX200A at 10, 5, and 1 mg/Kg).

To assess whether ADC INX200A confers improved efficacy, we compared the response to various Dex doses to an equivalent Dex payload from the ADC (INX200A at 0.2 mg/Kg free Dex = 10 mg/Kg , INX200A at 0.02 mg/Kg free Dex = 1 mg/Kg). As can be seen from the data in Figure 7, free Dex loses effectiveness in controlling cytokine responses at 0.02 mg/Kg, while INX200A at 1 mg/Kg remains effective. More generally, the data show that:
Experiment 1
• In female hVISTA KI mice, INX210 (INX210A) when conjugated to Dex showed efficacy in controlling ConA-induced G-CSF responses.
Experiment 2
• Male hVISTA KI mice show a more consistent response to ConA injury.
• INX210 (INX210A) when conjugated to Dex showed efficacy in controlling ConA-induced cytokine responses. Naked antibodies had no efficacy.
• INX210A administered at 10 mg/Kg delivers approximately 0.2 mg/Kg of Dex. The efficacy observed with INX210A was comparable to that of free Dex at 0.2 mg/Kg.
Experiment 3
Dose-response experiments show improved/increased efficacy when Dex payload is delivered via ADC INX200A, free Dex at 0.02 mg/Kg has no efficacy but via ADC The molar equivalents delivered indicate high potency.

Conclusion It is shown that anti-VISTA antibody (INX210) (INX210A) when conjugated to Dex can prevent ConA-induced inflammation more efficiently or better than free Dex at equimolar doses of Dex. Unconjugated INX210 does not have any effect. We also conjugated Dex to the anti-VISTA antibody INX200, as it is shown that free Dex at 0.02 mg/Kg has no efficacy while the molar equivalent delivered via ADC has high potency. is shown to have improved Dex delivery.

Example 3: Synthesis of Exemplary Steroid Payloads and Antibody-Drug Conjugates Procedures for the Synthesis of Steroid Payloads and Conjugates This example demonstrates the attachment of novel steroids according to the present invention, steroid linker conjugates to antibodies. the conjugate and the antibody, optionally an anti-VISTA antibody that binds human VISTA at physiological pH and has a short pK; or to an immune cell, typically a linker and/or a bifunctional or trifunctional group attached to an antibody or antibody fragment that targets another antigen that is selectively expressed on human immune cells. The synthesis of antibody drug conjugates (ADCs) containing these steroids is described.

As mentioned above, these steroids generally include glucocorticoid agonist compounds and will possess the previously disclosed structure of Formula I, II, or III. Exemplary compounds of Formulas I, II, and III are shown in Figures 118A-O. Synthesis of these and other exemplary compounds are described herein.

General Procedures The following general procedures were used for liquid chromatography (preparation or analysis) and nuclear magnetic resonance.

Liquid Chromatography Unless otherwise stated, the following conditions were used for high pressure liquid chromatography (HPLC) purification or for liquid chromatography mass spectrometry (LC-MS).

LCMS method A
Sample analysis by this method was performed on an Agilent 1260 LCMS-4-QUAD system using a 50 x 2 mm Onyx™ monolithic C18 LC column. Using a gradient of 5-95% A in B over 6 minutes, where A = 0.05% AcOH/ACN in water (95:5 v/v) and B = 0.05% AcOH in ACN. The sample was run.

LCMS method B
Sample analysis by this method was performed on a Waters Acquity LCMS-5-SQD system using a Kinetex® 1.7 μm C18 100 Å, LC column 50×2.1 mm. Samples were run using a gradient of 10-95% A in B over 2.5 minutes, where A = 0.02% formic acid in water and B = 0.05% formic acid in ACN.
Below is the LCMS method used for analysis of the final target.
LCMS method 1:
Column details: X-BRIDGE BEH 2.1 x 50mm 2.5μm
Machine details: Water Acquity UPLC-H class equipped with PDA and Acquity SQ detector, column temperature: 35 °C, autosampler temperature: 5 °C, mobile phase A: 0.1% formic acid in Milli-Q water (pH = 2.70), Mobile phase B: 0.1% formic acid in Milli-Q water: acetonitrile (10:90).
Mobile phase gradient details: T = 0 min (97% A, 3% B) flow rate: 0.8 mL/min, T = 0.75 min (97% A, 3% B) flow rate: 0.8 mL/min, T = Gradient (2% A, 98% B) up to 2.7 minutes Flow rate: 0.8 mL/min, T = Gradient (0% A, 100% B) up to 3 minutes Flow rate: 1 mL/min, T = 3 .5 min (0% A, 100% B) Flow rate: 1 mL/min, T = 3. Gradient (97% A, 3% B) flow rate to 51 min: 0.8 mL/min, T = 4 min. (97% A, 3% B), flow rate: 0.8 mL/min, flow rate: 0.8 mL/min, electrophoresis time: 4 minutes, UV detection method: PDA.
Mass parameters:
Probe: ESI, mode of ionization: positive and negative, cone voltage: 30V and 10V, capillary voltage: 3.0KV, extractor voltage: 1V, Rf lens: 0.1V, source temperature: 120°C, desolvation Japanese temperature: 400°C. Cone gas flow rate: 100 L/hour, desolvation gas flow rate: 800 L/hour.
LCMS method 2:
Column details: Xtimate C18 4.6 x 150mm 5μm
Machine details: Waters 996 photodiode array detector equipped with Waters micromass ZQ detector, column temperature: 35°C, autosampler temperature: 15°C, mobile phase A: 5mM ammonium acetate in Milli-Q water and 0.1 % formic acid (pH = 3.50), mobile phase B: methanol mobile phase gradient details: T = 0 min (90% A, 10% B), T = 7.0 min (10% A, 90% B), T = 9.0 min (0% A, 100% B), gradient to T = 14.00 min (0% A, 100% B), T = 14.01 min (90% A, 10% B) , end of electrophoresis at T=17 minutes (90% A, 10% B), flow rate: 1.0 mL/min, electrophoresis time: 17 minutes, UV detection method: PDA.
Mass parameters:
Probe: ESI, mode of ionization: positive and negative, cone voltage: 30 and 10V, capillary voltage: 3.0KV, extractor voltage: 2V, Rf lens: 0.1V, source temperature: 120 °C, probe Temperature: 400°C, cone gas flow rate: 100 L/hour, desolvation gas flow rate: 800 L/hour.
LCMS method 3:
Column details: Sunfire C18 150 x 4.6mm, 3.5μm
Machine details: Agilent 1260 Infinity-II and G6125C (LC/MSD) mass detector, column temperature: 35°C, autosampler temperature: 15°C, mobile phase A: 5mM ammonium acetate and 0.1% in Milli-Q water. Formic acid (pH = 3.50), mobile phase B: methanol Mobile phase gradient details: T = 0 min (90% A, 10% B), T = 7.0 min (10% A, 90% B), T = 9.0 min (0% A, 100% B), T = gradient to 14.00 min (0% A, 100% B), T = 14.01 min (90% A, 10% B), End of electrophoresis at T=17 minutes (90% A, 10% B), flow rate: 1.0 mL/min, electrophoresis time: 17 minutes, UV detection method: PDA.
Mass parameters:
Probe: MMI, mode of ionization: (ESI) positive and negative, fragment voltage: 30V and 70V, capillary voltage: 3000V, source gas temperature: 325°C, vaporizer temperature: 225°C, gas flow rate: 12L / Spray, atomizer: 50.
HPLC method 1:
Column details: Sunfire C18 (150mm x 4.6mm), 3.5μm
Machine details: Agilent Technologies. 1260 series, Infinity-II with PDA detector, column temperature: 35 °C, autosampler temperature: 15 °C, mobile phase A: 0.05% trifluoroacetic acid in Milli-Q water (pH = 2.2), mobile Phase B: Acetonitrile.
Mobile phase gradient details: T = 0 min (90% A, 10% B) flow rate: 1.0 mL/min, T = 7.0 min (10% A, 90% B) flow rate: 1.0 mL/min, T = Gradient (00% A, 100% B) up to 9.0 min Flow rate: 1.0 mL/min, T = Gradient (00% A, 100% B) up to 14 min Flow rate: 1.0 mL/min, T = 14.01 minutes (90% A, 10% B) flow rate: 1 mL/min, end of electrophoresis at T = 17 minutes (90% A, 10% B) flow rate: 1.0 mL/min, flow rate: 1.0 mL /min, electrophoresis time: 17 minutes, UV detection method: PDA.
HPLC method 2
Column details: Atlantis C18 (150 mm x 4.6 mm), 5.0 μm or Welch C18 (150 mm x 4.6 mm), 5.0 μm
Machine details: Waters Alliance e2695 with 2998 PDA detector, column temperature: 35 °C, autosampler temperature: 15 °C, mobile phase A: 0.1% ammonia in Milli-Q water (pH = 10.5), mobile phase B: Acetonitrile.
Mobile phase gradient details: T = 0 min (90% A, 10% B) flow rate: 1.0 mL/min, T = 7.0 min (10% A, 90% B) flow rate: 1.0 mL/min, T = Gradient (00% A, 100% B) up to 9.0 min Flow rate: 1.0 mL/min, T = Gradient (00% A, 100% B) up to 14 min Flow rate: 1.0 mL/min, T = 14.01 minutes (90% A, 10% B) flow rate: 1 mL/min, end of electrophoresis at T = 17 minutes (90% A, 10% B) flow rate: 1.0 mL/min, flow rate: 1.0 mL /min, electrophoresis time: 17 minutes, UV detection method: PDA.
HPLC details: Waters Alliance e2695 with 2998 PDA detector, column details: Atlantis C18 (150 mm x 4.6 mm), 5.0 μm or Welch C18 (150 mm x 4.6 mm), 5.0 μm, mobile phase A: Milli-Q in water 0.1% ammonia (pH = 10.5), mobile phase B: acetonitrile, flow rate: 1.0 mL/min, running time: 17 minutes

NMR
The following conditions were used to obtain proton nuclear magnetic resonance (NMR) spectra. NMR spectra were recorded on ^{a 1} H NMR (400 MHz) Bruker Advancer-III HD FT-NMR spectrophotometer (Bruker, USA). Crude NMR data was analyzed using Topspin 3.6.3 software.

Chemical shifts are reported in parts per million (ppm) downfield from the TMS position as estimated by deuterated NMR solvents. Apparent multiplicity is reported as singlet-s, doublet-d, triplet-t, quartet-q, or multiplet-m. Peaks exhibiting broadening are further expressed as br. Integration is an approximation. It should be mentioned that the integrated intensity, peak shape, chemical shift and coupling constant may depend on the solvent, concentration, temperature, pH and other factors.

Experimental Details All reactions were carried out under a dry nitrogen atmosphere unless otherwise stated. All major chemicals were used as received. All other commercially available materials such as solvents, reagents, and catalysts were used without further purification. The reaction was monitored by thin layer chromatography (TLC) using pre-coated Merck silica gel 60F254 aluminum sheets (Merck, Germany). Visualization of the TLC plate was achieved using ultraviolet light, ninhydrin spray, and iodine vapor. Column chromatographic separations were carried out using 230-400 mesh, 100-200 mesh, and 60-120 mesh silica gel or C18 silica as the stationary phase using the appropriate mobile phase.

（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ Ｊ．ａ）の合成：

手順：
丸底フラスコに、Ｆｍｏｃ－Ｇｌｙ－ＯＳｕ（１．０ｇ、２．５３５ｍｍｏｌ、１．０当量）、Ｈ－Ｇｌｕ（ＯｔＢｕ）－ＯＨ（０．６１８３ｇ、３．０４３ｍｍｏｌ、１．２当量）、及び重炭酸ナトリウム（０．４２６０ｇ、５．０７ｍｍｏｌ、２．０当量）を充填した。水及び１，４－ジオキサン（１：１、２６ｍＬ）の溶液を添加し、混合物を室温で一晩撹拌させた。出発物質の消費をＬＣＭＳによって確認し、溶媒を還元し、ジオキサンを除去したが、水を残した。次いで、混合物をｐＨ２～３まで酸性化し、分液漏斗に添加し、５：１の酢酸イソプロピル／イソプロパノール（３×１００ｍＬ）で抽出した。組み合わせた有機物をＮａ_２ＳＯ_４上で乾燥させ、濾過し、還元し、Ｉｓｃｏ Ｃ１８ Ａｑ １００ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として８２％収率で０．９９８２ｇのＩＮＸ Ｊ．ａを得た。ＬＣＭＳ方法Ｂ（ＥＳＩ＋）：Ｃ_２６Ｈ_３１Ｎ_２Ｏ_７［Ｍ＋Ｈ］^＋は、４８３．２１を必要とし、１．１４分における実測値４８３．２５である。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho Synthesis reaction scheme of [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benyl)phenyl)amino)-5-oxopentanoic acid (INX J)

(S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentanoic acid (INX J.a) Synthesis:

procedure:
In a round bottom flask, Fmoc-Gly-OSu (1.0 g, 2.535 mmol, 1.0 eq.), H-Glu(OtBu)-OH (0.6183 g, 3.043 mmol, 1.2 eq.), and Sodium carbonate (0.4260g, 5.07mmol, 2.0eq) was charged. A solution of water and 1,4-dioxane (1:1, 26 mL) was added and the mixture was allowed to stir at room temperature overnight. Consumption of starting material was confirmed by LCMS and the solvent was reduced and dioxane was removed but water remained. The mixture was then acidified to pH 2-3, added to a separatory funnel, and extracted with 5:1 isopropyl acetate/isopropanol (3 x 100 mL). The combined organics were dried over Na ₂ SO ₄ , filtered, reduced and loaded onto an Isco C18 Aq 100 g reverse phase column with 0-100% acetonitrile in H ₂ O (0.05% AcOH additive). (0.05% AcOH additive) mobile phase. Fractions containing pure product were combined, frozen, and lyophilized to yield 0.9982 g of INX J.I. in 82% yield as a white solid. I got a. LCMS Method B (ESI+): _C26H31N2O7 [M+H] ⁺ requires _483.21 , _found 483.25 _at 1.14 min.

手順：
丸底フラスコにアルゴンを戻して充填し、（３－（４，４，５，５－テトラメチル－１，３，２－ジオキサボロラン－２－イル）フェニル）カルバミン酸ｔｅｒｔ－ブチル（４．２７６５ｇ、１３．４０ｍｍｏｌ、１．０当量）、４－ブロモメチルベンズアルデヒド（４．０ｇ、２０．１ｍｍｏｌ、１．５当量）、炭酸カリウム（９．２５９４ｇ、６７．０ｍｍｏｌ、５．０当量）、及び［１．１′－ビス（ジフェニルホスフィノ）フェロセン］ジクロロパラジウム－ジクロロロメタン複合体（０．３８４１ｇ、０．４６９ｍｍｏｌ、０．０３５当量）を充填した。無水ＴＨＦ（８４ｍＬ）をフラスコに添加し、次いで、フラスコに還流凝縮器を装備し、８０℃まで１６時間加熱した。出発材料消費をＬＣＭＳによって確認し、次いで、混合物を冷却し、水（２００ｍＬ）で希釈し、分液漏斗に添加し、ＥｔＯＡｃ（３×１００ｍＬ）で抽出した。組み合わせた有機物をＮａ_２ＳＯ_４上で乾燥させ、濾過し、還元し、Ｉｓｃｏ Ｒｆ Ｇｏｌｄ ８０ｇ ＳｉＯ_２カラム上に装填し、ヘキサン中の０～１００％ＥｔＯＡｃの移動相で溶出した。純粋な生成物を含有する画分を組み合わせて還元し、透明な油として８５％収率で３．５２９ｇの化合物ＩＮＸ Ｊ－１を得て、これを減圧からの除去後に一晩結晶化させた。ＬＣＭＳ方法Ａ（ＥＳＩ－）：Ｃ_１９Ｈ_２０ＮＯ_３［Ｍ－Ｈ］^－は、３１０．１５を必要とし、３．０８０分における実測値３１０．１である。 Synthesis of tert-butyl (3-(4-formylbenzyl)phenyl)carbamate (INX J-1):

procedure:
Backfill the round bottom flask with argon and add tert-butyl (3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate (4.2765 g, 13.40 mmol, 1.0 eq), 4-bromomethylbenzaldehyde (4.0 g, 20.1 mmol, 1.5 eq), potassium carbonate (9.2594 g, 67.0 mmol, 5.0 eq), and [1 .1'-bis(diphenylphosphino)ferrocene]dichloropalladium-dichloromethane complex (0.3841 g, 0.469 mmol, 0.035 equivalents) was charged. Anhydrous THF (84 mL) was added to the flask, which was then equipped with a reflux condenser and heated to 80° C. for 16 hours. Starting material consumption was confirmed by LCMS, then the mixture was cooled, diluted with water (200 mL), added to a separatory funnel, and extracted with EtOAc (3 x 100 mL). The combined organics were dried over Na ₂ SO ₄ , filtered, reduced and loaded onto an Isco Rf Gold 80 g SiO ₂ column and eluted with a mobile phase of 0-100% EtOAc in hexanes. The fractions containing pure product were combined and reduced to give 3.529 g of compound INX J-1 in 85% yield as a clear oil, which crystallized overnight after removal from vacuum. . LCMS Method A (ESI-): C ₁₉ H ₂₀ NO ₃ [MH] ^- requires 310.15, found 310.1 at 3.080 min.

手順：
丸底フラスコに、１６－α－ヒドロキシプレドニゾロン（３．３０ｇ、８．７６５ｍｍｏｌ、１．０当量）、アルデヒドＩＮＸ Ｊ－１（３．００２３ｇ、９．６４１ｍｍｏｌ、１．１当量）、及びＭｇＳＯ_４（３．１６５９ｇ、２６．２９ｍｍｏｌ、３．０当量）を充填した。固体をアセトニトリル（８８ｍＬ）中に懸濁し、混合物を０℃まで冷却すると、トリフルオロメタンスルホン酸（３．９ｍＬ、４３．８３ｍｍｏｌ、５．０当量）を滴加した。１０～２０分後、反応物がピンク色に変わり、出発物質が１時間後に完全に消費された。溶媒を還元し、粗製物を２つのバッチで精製し、各々、Ｉｓｃｏ Ｃ１８ Ａｑ ２７５ｇ逆相カラムに装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の５～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として５０％収率で２．５０ｇのＩＮＸ Ｊ－２を得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_３５Ｈ_４０ＮＯ_６［Ｍ＋Ｈ］^＋は、５７０．２８を必要とし、２．５７２分における実測値５７０．３である。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-(3-aminobenzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a -dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d Synthesis of [1,3]dioxol-4-one (INX J-2):

procedure:
In a round bottom flask, 16-α-hydroxyprednisolone (3.30 g, 8.765 mmol, 1.0 eq.), aldehyde INX J-1 (3.0023 g, 9.641 mmol, 1.1 eq.), and MgSO ₄ ( 3.1659g, 26.29mmol, 3.0eq) was charged. The solid was suspended in acetonitrile (88 mL) and the mixture was cooled to 0° C. and trifluoromethanesulfonic acid (3.9 mL, 43.83 mmol, 5.0 eq.) was added dropwise. After 10-20 minutes, the reaction turned pink and the starting material was completely consumed after 1 hour. The solvent was reduced and the crude was purified in two batches, each loaded onto an Isco C18 Aq 275g reverse phase column, containing 5-100% acetonitrile (0.0% AcOH additive) in H ₂ O (0.05% AcOH additive). Elution was carried out with a mobile phase of 0.05% AcOH (additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 2.50 g of INX J-2 as a white solid in 50% yield. LCMS Method A (ESI+): _C35H40NO6 [M+H] ⁺ requires _{570.28 ,} found 570.3 at 2.572 minutes.

手順：
ＤＭＦ（２．３ｍＬ）を、ビスアミノ酸ＩＮＸ Ｊ．ａ（０．３０７４ｇ、０．６３７２ｍｍｏｌ、１．１当量）を充填した丸底フラスコに添加した。次いで、アニリンＩＮＸ Ｊ－２（０．３３０ｇ、０．５７９ｍｍｏｌ、１．０当量）、続いて、トリエチルアミン（０．２４ｍＬ、１．７３ｍｍｏｌ、３．０当量）を添加した。溶液を０℃まで冷却し、ＤＭＦ中の次いで、５０％プロパンホスホン酸無水物の溶液（０．７０ｍＬ、１．１５８６ｍｍｏｌ、２．０当量）を添加した。混合物を室温まで加温しながら１６時間撹拌させた。反応完了がＬＣＭＳによって確認されると、粗混合物をＩｓｃｏ Ｃ１８ Ａｑ ５０ｇ逆相カラム上に直接装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として３３％収率で０．２００ｇのＩＮＸ Ｊ－３を得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_６１Ｈ_６８Ｎ_３Ｏ_１２［Ｍ＋Ｈ］^＋は、１０３４．４７を必要とし、３．０７３分における実測値１０３４．４である。 (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a , 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5 -Synthesis of tert-butyl oxopentanoate (INX J-3):

procedure:
DMF (2.3 mL) was mixed with bisamino acid INX J. a (0.3074 g, 0.6372 mmol, 1.1 eq.) was added to a round bottom flask filled with a. Aniline INX J-2 (0.330 g, 0.579 mmol, 1.0 eq.) was then added followed by triethylamine (0.24 mL, 1.73 mmol, 3.0 eq.). The solution was cooled to 0° C. and a solution of 50% propanephosphonic anhydride (0.70 mL, 1.1586 mmol, 2.0 eq.) in DMF was then added. The mixture was allowed to stir for 16 hours while warming to room temperature. Once reaction completion was confirmed by LCMS, the crude mixture was loaded directly onto an Isco C18 Aq 50 g reverse phase column and 0-100% acetonitrile (0.05%) in H ₂ O (0.05% AcOH additive). It was eluted with a mobile phase of (AcOH additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 0.200 g of INX J-3 as a white solid in 33% yield. LCMS Method A (ESI+): C ₆₁ H ₆₈ N ₃ O ₁₂ [M+H] ⁺ requires 1034.47, found 1034.4 at 3.073 min.

手順：
バイアルに、化合物ＩＮＸ Ｊ－３（０．０８０ｇ、０．０７７４ｍｍｏｌ、１．０当量）を充填し、次いで、これをアセトニトリル（０．５０ｍＬ）及びピペリジン（６２μＬ）に溶解させた。混合物を、全ての出発物質が脱保護されるまで３０分間撹拌させた。溶媒を還元し、粗製物をＤＭＳＯ中で希釈し、Ｉｓｃｏ Ｃ１８ Ａｑ １５．５ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、透明な油として６３％収率で０．０４２３ｇのＩＮＸ Ｊ－４・ＡｃＯＨを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_４６Ｈ_５８Ｎ_３Ｏ_１０［Ｍ＋Ｈ］^＋は、８１２．４０を必要とし、２．６３８分における実測値８１２．４である。 tert-Butyl (S)-4-(2-aminoacetamido)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2 ',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5-oxopentanoate AcOH salt (INX J-4) Synthesis:

procedure:
A vial was charged with compound INX J-3 (0.080 g, 0.0774 mmol, 1.0 eq.), which was then dissolved in acetonitrile (0.50 mL) and piperidine (62 μL). The mixture was allowed to stir for 30 minutes until all starting material was deprotected. The solvent was reduced and the crude was diluted in DMSO and loaded onto an Isco C18 Aq 15.5 g reverse phase column, 0-100% acetonitrile (0.5% AcOH additive) in H ₂ O (0.05% AcOH additive). Elution was carried out with a mobile phase of 0.05% AcOH (additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 0.0423 g of INX J-4.AcOH as a clear oil in 63% yield. LCMS Method A (ESI+): C ₄₆ H ₅₈ N ₃ O ₁₀ [M+H] ⁺ requires 812.40, found 812.4 at 2.638 min.

手順：
バイアルに、２－ブロモ酢酸（０．００９２ｇ、０．０６６５ｍｍｏｌ、２．１当量）及びＤＭＦ（０．３３ｍＬ）を充填した。Ｎ－エトキシカルボニル－２－エトキシ－１，２－ジヒドロキノリン（０．０１５６ｇ、０．０６３２ｍｍｏｌ、２．０当量）を添加し、混合物を約９０分間撹拌した。次いで、アミンＩＮＸ Ｊ－４・ＡｃＯＨ（０．０２７０ｇ、０．０３０９ｍｍｏｌ、１．０当量）を、重炭酸ナトリウム（０．０１４０ｇ、０．１６６５ｍｍｏｌ、５．４当量）とともに溶液に添加し、混合物を２時間（全てのＩＮＸ Ｊ－４が消費されるまで）撹拌させた。反応完了がＬＣＭＳによって確認されると、粗混合物をＩｓｃｏ Ｃ１８ Ａｑ ５．５ｇ逆相カラム上に直接装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として３５％収率で０．０１００ｇのＩＮＸ Ｊ－５を得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_４８Ｈ_５９ＢｒＮ_３Ｏ_１１［Ｍ＋Ｈ］^＋は、９３２．３３を必要とし、２．９２６分における実測値９３２．２である。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-tert-butyl-5-oxopentanoate (INX J-5 ) composition:

procedure:
A vial was charged with 2-bromoacetic acid (0.0092 g, 0.0665 mmol, 2.1 eq.) and DMF (0.33 mL). N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (0.0156 g, 0.0632 mmol, 2.0 eq.) was added and the mixture was stirred for about 90 minutes. Amine INX J-4 AcOH (0.0270 g, 0.0309 mmol, 1.0 eq.) was then added to the solution along with sodium bicarbonate (0.0140 g, 0.1665 mmol, 5.4 eq.) and the mixture Allow to stir for 2 hours (until all INX J-4 is consumed). Once reaction completion was confirmed by LCMS, the crude mixture was loaded directly onto an Isco C18 Aq 5.5 g reverse phase column and 0-100% acetonitrile (0.5% AcOH additive) in H ₂ O (0.05% AcOH additive). Elution was carried out with a mobile phase of 0.05% AcOH (additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 0.0100 g of INX J-5 as a white solid in 35% yield. LCMS Method A (ESI+): _{C48H59BrN3O11} [ _M + _H ] ⁺ requires _932.33 , found 932.2 at 2.926 min.

手順：
バイアルに、ｔｅｒｔ－ブチルエステルＩＮＸ Ｊ－５（０．０１０ｇ、０．０１０７２ｍｍｏｌ、１．０当量）を充填し、これをＤＣＭ中の５０％ＴＦＡの溶液（０．２００ｍＬ）に溶解させ、１時間撹拌させた。反応完了がＬＣＭＳによって確認されると、溶媒を除去し、残渣をＤＭＳＯに溶解させ、Ｉｓｃｏ Ｃ１８ Ａｑ ５．５ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として３５％収率で０．００３３ｇのＩＮＸ Ｊを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_４４Ｈ_５１ＢｒＮ_３Ｏ_１１［Ｍ＋Ｈ］^＋は、８７６．２６を必要とし、２．５２４分における実測値８７７．２である。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho Synthesis of [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benyl)phenyl)amino)-5-oxopentanoic acid (INX J):

procedure:
A vial was charged with tert-butyl ester INX J-5 (0.010 g, 0.01072 mmol, 1.0 equiv.), which was dissolved in a solution of 50% TFA in DCM (0.200 mL) for 1 hour. It was stirred. Once reaction completion was confirmed by LCMS, the solvent was removed and the residue was dissolved in DMSO and loaded onto an Isco C18 Aq 5.5 g reverse phase column in H ₂ O (0.05% AcOH additive). Elution was with a mobile phase of 0-100% acetonitrile (0.05% AcOH additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 0.0033 g of INX J as a white solid in 35% yield. LCMS Method A (ESI+): C ₄₄ H ₅₁ BrN ₃ O ₁₁ [M+H] ⁺ requires 876.26, found 877.2 at 2.524 min.

（Ｓ）－５－（ｔｅｒｔ－ブトキシ）－２－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）アセトアミド）－５－オキソペンタン酸（Ｂｏｃ－Ｇｌｙ－Ｇｌｕ（ＯｔＢｕ）－ＯＨ）の合成：

手順：
丸底フラスコに、Ｂｏｃ－Ｇｌｙ－ＯＳｕ（１２．０ｇ、４４．０７ｍｍｏｌ、１．０当量）、Ｈ－Ｇｌｕ（ＯｔＢｕ）－ＯＨ（９．８５２４ｇ、４８．４７ｍｍｏｌ、１．１当量）、及び重炭酸ナトリウム（７．４０４０ｇ、８８．１４ｍｍｏｌ、２．０当量）を充填した。水及び１，４－ジオキサン（１：１、２２０ｍＬ）の溶液を添加し、混合物を室温で一晩撹拌させた。出発物質の消費をＬＣＭＳによって確認し、溶媒を還元し、ジオキサンを除去したが、水を残した。次いで、混合物をｐＨ２～３まで酸性化し、沈殿物を形成し、これを次いで、濾過し、凍結乾燥剤上で乾燥させて、白色の固体として８８％収率で１４．０１５２ｇのＢｏｃ－Ｇｌｙ－Ｇｌｕ（ＯｔＢｕ）－ＯＨを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_１６Ｈ_２９Ｎ_２Ｏ_７［Ｍ＋Ｈ］^＋は、３６１．１９を必要とし、２．１２２分における実測値３６１．２である。 S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -6a,8a-dimethyl-4-oxo-8b-(2-(phosphonoxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H- Synthesis reaction of naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5-oxopentanoic acid (INX L) scheme

Synthesis of (S)-5-(tert-butoxy)-2-(2-((tert-butoxycarbonyl)amino)acetamido)-5-oxopentanoic acid (Boc-Gly-Glu(OtBu)-OH):

procedure:
In a round bottom flask, Boc-Gly-OSu (12.0 g, 44.07 mmol, 1.0 eq.), H-Glu(OtBu)-OH (9.8524 g, 48.47 mmol, 1.1 eq.), and weight Sodium carbonate (7.4040g, 88.14mmol, 2.0eq) was charged. A solution of water and 1,4-dioxane (1:1, 220 mL) was added and the mixture was allowed to stir at room temperature overnight. Consumption of starting material was confirmed by LCMS and the solvent was reduced and dioxane was removed but water remained. The mixture was then acidified to pH 2-3 to form a precipitate, which was then filtered and dried on a lyophilizer to yield 14.0152 g of Boc-Gly- in 88% yield as a white solid. Glu(OtBu)-OH was obtained. _LCMS Method A (ESI+): _C16H29N2O7 [M+H] ⁺ requires _361.19 , _found 361.2 at 2.122 minutes.

手順：
不活性雰囲気下のオーブンで乾燥させたバイアルに、アミンＩＮＸ Ｊ－２（０．８２００ｇ、２．６３ｍｍｏｌ、１．０当量）、Ｂｏｃ－Ｇｌｙ－Ｇｌｕ（ＯｔＢｕ）－ＯＨ（２．５９１６ｇ、７．１９７ｍｍｏｌ、２．７３当量）、（（７－アザベンゾトリアゾール－１－イルオキシ）トリピロリジノホスホニウムヘキサフルオロリンホスフェート）（２．２５１５ｇ、４．３１８ｍｍｏｌ、１．６４当量）、及びＤＭＦ（１５ｍＬ）を充填した。次に、Ｎ，Ｎ－ジイソプロピルエチルアミン（１．５ｍＬ、８．６３６ｍｍｏｌ、３．３当量）を添加し、混合物を全てのアミンが消費されるまで１時間撹拌させた。次いで、粗溶液を、Ｉｓｃｏ Ｃ１８ Ａｑ １００ｇ逆相カラムに直接添加し、Ｈ_２Ｏ（０．０５％ＴＦＡ添加剤）中の０～１００％アセトニトリル（０．０５％ＴＦＡ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として３４％収率で０．４４０４ｇのＩＮＸ Ｌ－１を得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_５１Ｈ_６６Ｎ_３Ｏ_１２［Ｍ＋Ｈ］^＋は９１２．４６を必要とし、２．５２４分における実測値９１２．４である。 (S)-4-(2-((tert-butoxycarbonyl)amino)acetamide)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenylamino)-5 -Synthesis of tert-butyl oxopentanoate (INX-1):

procedure:
Amine INX J-2 (0.8200 g, 2.63 mmol, 1.0 eq.), Boc-Gly-Glu(OtBu)-OH (2.5916 g, 7.0 eq.) were added to an oven-dried vial under an inert atmosphere. (197 mmol, 2.73 eq.), ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluoroline phosphate) (2.2515 g, 4.318 mmol, 1.64 eq.), and DMF (15 mL). Filled. Next, N,N-diisopropylethylamine (1.5 mL, 8.636 mmol, 3.3 eq.) was added and the mixture was allowed to stir for 1 hour until all the amine was consumed. The crude solution was then applied directly to an Isco C18 Aq 100 g reverse phase column with a mobile phase of 0-100% acetonitrile (0.05% TFA additive) in H ₂ O (0.05% TFA additive). It eluted. Fractions containing pure product were combined, frozen, and lyophilized to yield 0.4404 g of INX L-1 as a white solid in 34% yield. LCMS Method A (ESI+): C ₅₁ H ₆₆ N ₃ O ₁₂ [M+H] ⁺ requires 912.46, found 912.4 at 2.524 min.

手順：
不活性雰囲気下のオーブンで乾燥させたバイアルに、ｔｅｒｔ－ブチルエステルＩＮＸ Ｌ－１（０．２００ｇ、０．２２０ｍｍｏｌ、１．０当量）及びＤＭＦ（０．５０ｍＬ）を充填した。次に、１－Ｈテトラゾール（０．１５４０ｇ、２．２０ｍｍｏｌ、１０当量）及びジ－ｔｅｒｔ－ブチルＮ，Ｎ－ジエチルホスホラミダイト（１．３１１ｇ、５．２６５ｍｍｏｌ、２４．０当量）を添加し、混合物を７２時間撹拌させて、９０％変換を達成した。過酸化水素（２ｍＬ）を添加し、Ｉｓｃｏ Ｃ１８ Ａｑ ５０ｇ逆相カラムに充填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出する前に、混合物を１時間撹拌させた。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として４９％収率で０．１２０ｇのＩＮＸ Ｌ－２を得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_５９Ｈ_８３Ｎ_３Ｏ_１５Ｐ［Ｍ＋Ｈ］^＋は、１１０４．５５を必要とし、３．８９４分における実測値１１０４．５である。 (S)-4-(2-((tert-butoxycarbonyl)amino)acetamide)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenylamino)-5 -Synthesis of tert-butyl oxopentanoate (INX L-2):

procedure:
An oven-dried vial under an inert atmosphere was charged with tert-butyl ester INX L-1 (0.200 g, 0.220 mmol, 1.0 eq.) and DMF (0.50 mL). Next, 1-H tetrazole (0.1540 g, 2.20 mmol, 10 eq.) and di-tert-butyl N,N-diethylphosphoramidite (1.311 g, 5.265 mmol, 24.0 eq.) were added. , the mixture was allowed to stir for 72 hours to achieve 90% conversion. Hydrogen peroxide (2 mL) was added and packed into an Isco C18 Aq 50 g reverse phase column with 0-100% acetonitrile (0.05% AcOH additive) in H ₂ O (0.05% AcOH additive). The mixture was allowed to stir for 1 hour before elution with the mobile phase. Fractions containing pure product were combined, frozen, and lyophilized to yield 0.120 g of INX L-2 as a white solid in 49% yield. LCMS Method A (ESI+): _C59H83N3O15P [ _M +H] ⁺ requires _1104.55 , found 1104.5 at 3.894 _min .

手順：
丸底フラスコに、ｔｅｒｔ－ブチルエステルＩＮＸ Ｌ－２（０．７７２ｇ、０．７ｍｍｏｌ、１．０当量）、ＤＣＭ（１０ｍＬ）、トリフルオロ酢酸（５ｍＬ）、及びトリイソプロピルシラン（１．２ｍＬ）を充填した。混合物を室温で８時間撹拌させた。出発物質の消費をＬＣＭＳによって確認し、溶媒を還元した。結果として得られた残渣をＤＭＦ（４ｍＬ）に溶解させ、Ｉｓｃｏ Ｃ１８ Ａｑ １００ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．０５％ＴＦＡ添加剤）中の０～１００％アセトニトリル（０．０５％ＴＦＡ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として５４％収率で０．３９７６ｇのＩＮＸ Ｌ－３・ＴＦＡを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_４２Ｈ_５１Ｎ_３Ｏ_１３Ｐ［Ｍ＋Ｈ］^＋は、８３６．３を必要とし、２．０５３分における実測値８３６．３である。 (S)-4-(2-aminoacetamide)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-6a, 8a -dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2' , 1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5-oxopentanoic acid TFA salt (INX L-3):

procedure:
In a round bottom flask were added tert-butyl ester INX L-2 (0.772 g, 0.7 mmol, 1.0 eq.), DCM (10 mL), trifluoroacetic acid (5 mL), and triisopropylsilane (1.2 mL). Filled. The mixture was allowed to stir at room temperature for 8 hours. Consumption of starting material was confirmed by LCMS and solvent was reduced. The resulting residue was dissolved in DMF (4 mL) and loaded onto an Isco C18 Aq 100 g reverse phase column, 0-100% acetonitrile (0.05% TFA additive) in H ₂ O (0.05% TFA additive). % TFA additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 0.3976 g of INX L-3.TFA as a white solid in 54% yield. LCMS Method A (ESI+): _C42H51N3O13P [M+H] ⁺ requires _836.3 , _found 836.3 at 2.053 _minutes .

手順：
丸底フラスコに、２－ブロモ酢酸（０．０２５０ｇ、０．１８０ｍｍｏｌ、３．５当量）、ＤＭＦ（０．５０ｍＬ）、（７－アザベンゾトリアゾール－１－イルオキシ）トリピロリジノホスホニウムヘキサフルオロホスフェート（０．０４７０ｇ、０．０９０ｍｍｏｌ、１．７当量）、及びＮ，Ｎ－ジイソプロピルエチルアミン（０．０１５５ｇ、０．１２０ｍｍｏｌ、２．３当量）を充填した。別のバイアルに、アミンＩＮＸ Ｌ－３・ＴＦＡ（０．０５０ｇ、０．０５２ｍｍｏｌ、１．０当量）をＤＭＦ（２．０ｍＬ）中に溶解させ、ブロモ酢酸及びカップリング剤を含む容器に添加した。混合物を３０分間撹拌させ、出発物質の消費をＬＣＭＳによって確認した。粗混合物を、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相を用いた分取ＨＰＬＣによって精製した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として６０％収率で０．０３０ｇのＩＮＸ Ｌを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_４４Ｈ_５２ＢｒＮ_３Ｏ_１４Ｐ［Ｍ＋Ｈ］^＋は、９５６．７８を必要とし、２．３２３分における実測値９５６．２である。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonoxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H -Synthesis of naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5-oxopentanoic acid (INX L) :

procedure:
In a round bottom flask were added 2-bromoacetic acid (0.0250 g, 0.180 mmol, 3.5 eq.), DMF (0.50 mL), (7-azabenzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate ( 0.0470 g, 0.090 mmol, 1.7 eq.) and N,N-diisopropylethylamine (0.0155 g, 0.120 mmol, 2.3 eq.). In a separate vial, amine INX L-3 TFA (0.050 g, 0.052 mmol, 1.0 equiv.) was dissolved in DMF (2.0 mL) and added to the vessel containing the bromoacetic acid and coupling agent. . The mixture was allowed to stir for 30 minutes and consumption of starting material was confirmed by LCMS. The crude mixture was purified by preparative HPLC using a mobile phase of 0-100% acetonitrile (0.05% AcOH additive) in H ₂ O (0.05% AcOH additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 0.030 g of INXL as a white solid in 60% yield. LCMS Method A (ESI+): _{C44H52BrN3O14P} [ _M +H] ⁺ requires _956.78 , _found 956.2 at 2.323 minutes.

（Ｓ）－（１－（（４－（ヒドロキシメチル）フェニル）アミノ）－１－オキソプロパン－２－イル）カルバミン酸アリル（ＩＮＸ－ＳＭ－１－１）の合成：

手順：
不活性雰囲気下の丸底フラスコに、４－（ブロモメチル）ベンズアルデヒド（１．４６５ｇ、７．４０ｍｍｏｌ、１．２当量）、（５－（トリブチルスタニル）チアゾール－２－イル）カルバミン酸ｔｅｒｔ－ブチル（３．００ｇ、６．１０ｍｍｏｌ、１．０当量）、リン酸三カリウム（３．９０２ｇ、１８．４０ｍｍｏｌ、３．０当量）、及び（２－ジシクロヘキシルホスフィノ－２′，６′－ジメトキシビフェニル）［２－（２’－アミノ－１，１’－ビフェニル）］パラジウム（ＩＩ）メタンスルホネート（１．１４８ｇ、１．５０ｍｍｏｌ、２０ｍｏｌ％）を充填した。水（１０ｍＬ）及びＴＨＦ（１００ｍＬ）を脱気し、次いで、添加し、混合物を一晩還流させた。ＬＣＭＳを介して決定した完了時に、混合物を室温まで冷却し、還元し、Ｉｓｃｏ Ｃ１８ Ａｑ ４５０ｇ逆相カラム上に装填し、Ｈ_２Ｏ（１０ｍＭ ＮＨ_４ＯＡｃ添加剤）中の０～１００％アセトニトリル（１０ｍＭ ＮＨ_４ＯＡｃ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として５５％収率で１．０６４ｇのＩＮＸ－ＳＭ－１－１を得た。ＬＣＭＳ方法Ｂ（ＥＳＩ＋）：Ｃ_１１Ｈ_１１Ｎ_２ＯＳ［Ｍ－Ｂｏｃ＋Ｈ］^＋は、２１９．１０を必要とし、１．６６分で２１９．０４を見出した。 Synthesis reaction scheme of INX-SM-1 and INX N

Synthesis of allyl (S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)carbamate (INX-SM-1-1):

procedure:
In a round bottom flask under an inert atmosphere, 4-(bromomethyl)benzaldehyde (1.465 g, 7.40 mmol, 1.2 eq.), tert-butyl (5-(tributylstannyl)thiazol-2-yl)carbamate. (3.00 g, 6.10 mmol, 1.0 eq.), tripotassium phosphate (3.902 g, 18.40 mmol, 3.0 eq.), and (2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl) ) [2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate (1.148 g, 1.50 mmol, 20 mol%) was charged. Water (10 mL) and THF (100 mL) were degassed and then added and the mixture was refluxed overnight. Upon completion, as determined via LCMS, the mixture was cooled to room temperature, reduced and loaded onto an Isco _C18 Aq 450 g reverse phase column with _0-100 % acetonitrile ( Elution was with a mobile phase of 10 mM _NH4OAc (additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 1.064 g of INX-SM-1-1 as a white solid in 55% yield. LCMS Method B (ESI+): C ₁₁ H ₁₁ N ₂ OS[M-Boc+H] ⁺ required 219.10 and found 219.04 in 1.66 min.

手順：
丸底フラスコに、１６－α－ヒドロキシプレドニゾロン（１．１８３３ｇ、３．１４３ｍｍｏｌ、１．０当量）、アルデヒドＩＮＸ－ＳＭ－１－１（１．１０ｇ、３．４５８ｍｍｏｌ、１．１当量）、及びＭｇＳＯ_４（１．１３５５ｇ、９．４３１ｍｍｏｌ、３．０当量）を充填した。固体をアセトニトリル（３１ｍＬ）中に懸濁し、混合物を０℃まで冷却すると、トリフルオロメタンスルホン酸（１．４ｍＬ、１５．７１８ｍｍｏｌ、５．０当量）を滴加した。１０～２０分後、反応物がピンク色に変わり、出発物質が１時間後に消費された。溶媒を還元し、粗製物をＩｓｃｏ Ｃ１８ Ａｑ ２７５ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として５３％収率で１．０５９ｇのＩＮＸ－ＳＭ－１・ＡｃＯＨを得た。ＬＣＭＳ方法Ｂ（ＥＳＩ＋）：Ｃ_３２Ｈ_３７Ｎ_２Ｏ_６Ｓ［Ｍ＋Ｈ］^＋は、５７７．２３を必要とし、１．１０分で５７７．９３を見出した。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((2-aminothiazol-5-yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5] Synthesis of indeno[1,2-d][1,3]dioxol-4-one AcOH salt (INX-SM-1):

procedure:
In a round bottom flask, 16-α-hydroxyprednisolone (1.1833 g, 3.143 mmol, 1.0 eq.), aldehyde INX-SM-1-1 (1.10 g, 3.458 mmol, 1.1 eq.), and Charged with MgSO ₄ (1.1355g, 9.431mmol, 3.0eq). The solid was suspended in acetonitrile (31 mL) and the mixture was cooled to 0° C. and trifluoromethanesulfonic acid (1.4 mL, 15.718 mmol, 5.0 eq.) was added dropwise. After 10-20 minutes, the reaction turned pink and the starting material was consumed after 1 hour. The solvent was reduced and the crude was loaded onto an Isco C18 Aq 275 g reverse phase column, transferred from 0 to 100% acetonitrile (0.05% AcOH additive) in H ₂ O (0.05% AcOH additive). The phase eluted. Fractions containing pure product were combined, frozen, and lyophilized to yield 1.059 g of INX-SM-1·AcOH as a white solid in 53% yield. LCMS Method B (ESI+): _C32H37N2O6S [ _M +H] ⁺ required _577.23 and found 577.93 in 1.10 _min .

手順：
丸底フラスコに、ＩＮＸ－ＳＭ－１・ＡｃＯＨ（１．０００ｇ、１．５７ｍｍｏｌ、１．０当量）、Ｂｏｃ－Ｇｌｙ－Ｇｌｕ（ＯｔＢｕ）－ＯＨ（３．１２１２ｇ、８．６０７ｍｍｏｌ、５．５当量）、及びＰｙＡＯＰ（４．５２１０ｇ、８．６７８ｍｍｏｌ、５．５当量）を充填した。１：１のＤＣＭ／ＤＭＦの混合物（２２ｍＬの総体積）を添加し、続いて、ＤＩＰＥＡ（３．０ｍＬ、１７．３５６ｍｍｏｌ、１１．０当量）を添加し、混合物を５時間撹拌した。ＩＮＸ－ＳＭ－１の大部分が消費されると、溶媒を（ちょうどＤＭＦに）還元し、粗混合物をＩｓｃｏ Ｃ１８ Ａｑ ２７５ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の５～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として２８％収率で０．４０５０ｇのＩＮＸ Ｎ－１を得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_４８Ｈ_６３Ｎ_４Ｏ_１２Ｓ［Ｍ＋Ｈ］^＋は、９１９．４１を必要とし、３．０８９分で９１９．４を見出した。 (S)-4-(2-((tert-butoxycarbonyl)amino)acetamide)-5-((5-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)thiazol-2-yl)amino)-5-oxapentanoic acid tert -Synthesis of butyl (INX N-1):

procedure:
In a round bottom flask, INX-SM-1 AcOH (1.000 g, 1.57 mmol, 1.0 equivalent), Boc-Gly-Glu(OtBu)-OH (3.1212 g, 8.607 mmol, 5.5 equivalent) ), and PyAOP (4.5210 g, 8.678 mmol, 5.5 eq.). A 1:1 mixture of DCM/DMF (22 mL total volume) was added followed by DIPEA (3.0 mL, 17.356 mmol, 11.0 eq.) and the mixture was stirred for 5 hours. Once most of the INX-SM-1 was consumed, the solvent was reduced (just to DMF) and the crude mixture was loaded onto an Isco C18 Aq 275 g reverse phase column and washed with H ₂ O (0.05% AcOH additive). ) with a mobile phase of 5-100% acetonitrile (0.05% AcOH additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 0.4050 g of INX N-1 as a white solid in 28% yield. LCMS Method A (ESI+): _C48H63N4O12S [ _M +H] ⁺ required _919.41 and found 919.4 in 3.089 _minutes .

手順：
丸底フラスコに、ｔｅｒｔ－ブチルエステルＩＮＸ Ｎ－１（０．２００ｇ、０．２１７７ｍｍｏｌ、１．０当量）、ＭｅＣＮ（２．０ｍＬ）、トリフルオロ酢酸（２．０ｍＬ）、及びトリイソプロピルシラン（０．７０ｍＬ、３．２６６ｍｍｏｌ、１５．０当量）を充填した。混合物を室温で３時間撹拌させた。出発物質の消費をＬＣＭＳによって確認し、溶媒を還元した。結果として得られた残渣を、Ｉｓｃｏ Ｃ１８ Ａｑ ３０ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．１０％ＴＦＡ添加剤）中の０～１００％アセトニトリル（０．１０％ＴＦＡ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として５０％収率で０．０９５４ｇのＩＮＸ Ｎ－２・ＴＦＡを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_３９Ｈ_４７Ｎ_４Ｏ_１０Ｓ［Ｍ＋Ｈ］^＋は、７６３．２９を必要とし、１．７３２分で７６３．３を見出した。 (S)-4-(2-aminoacetamide)-5-((5-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)thiazol-2-yl)amino)-5-oxopentanoic acid TFA salt (INX N-2) Synthesis:

procedure:
In a round bottom flask, tert-butyl ester INX N-1 (0.200 g, 0.2177 mmol, 1.0 equiv.), MeCN (2.0 mL), trifluoroacetic acid (2.0 mL), and triisopropylsilane (0.0 .70 mL, 3.266 mmol, 15.0 eq.). The mixture was allowed to stir at room temperature for 3 hours. Consumption of starting material was confirmed by LCMS and solvent was reduced. The resulting residue was loaded onto an Isco C18 Aq 30 g reverse phase column and transferred from 0 to 100% acetonitrile (0.10% TFA additive) in H ₂ O (0.10% TFA additive). The phase eluted. Fractions containing pure product were combined, frozen, and lyophilized to yield 0.0954 g of INX N-2.TFA as a white solid in 50% yield. LCMS Method A (ESI+): _C39H47N4O10S [ _M +H] ⁺ required _763.29 and found 763.3 in 1.732 _min .

手順：
バイアルに、２－ブロモ酢酸（０．０１２７ｇ、０．０９１３ｍｍｏｌ、２．０当量）を充填し、これをＤＭＦ（０．５００ｍＬ）に溶解させた。Ｎ－エトキシカルボニル－２－エトキシ－１，２－ジヒドロキノリン（０．０２１５ｇ、０．０８６７ｍｍｏｌ、１．９当量）を添加し、混合物を９０分間撹拌させた。次いで、アミンＩＮＸ Ｎ－２・ＴＦＡ（０．０４０ｇ、０．０４５７ｍｍｏｌ、１．０当量）を重炭酸ナトリウム（０．０２３０ｇ、０．２７３９ｍｍｏｌ、６．０当量）とともに溶液に添加し、混合物を２時間（全てのＩＮＸ Ｎ－２が消費されるまで）撹拌させた。反応完了がＬＣＭＳによって確認されると、粗混合物をＩｓｃｏ Ｃ１８ Ａｑ １５．５ｇ逆相カラム上に直接装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、綿毛状の黄色の固体として２２％収率で０．００９１ｇのＩＮＸ Ｎを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_４１Ｈ_４８ＢｒＮ_４Ｏ_１１Ｓ［Ｍ＋Ｈ］^＋は、８８３．２１を必要とし、２．２４７分で８８３．２を見出した。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- -Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H- naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)thiazol-2-yl)amino)-5-oxopentanoic acid (INX N ) composition:

procedure:
A vial was charged with 2-bromoacetic acid (0.0127g, 0.0913mmol, 2.0eq), which was dissolved in DMF (0.500mL). N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (0.0215g, 0.0867mmol, 1.9eq) was added and the mixture was allowed to stir for 90 minutes. Amine INX N-2.TFA (0.040 g, 0.0457 mmol, 1.0 eq.) was then added to the solution along with sodium bicarbonate (0.0230 g, 0.2739 mmol, 6.0 eq.) and the mixture was Allow to stir for an hour (until all INX N-2 is consumed). Once reaction completion was confirmed by LCMS, the crude mixture was loaded directly onto an Isco C18 Aq 15.5 g reverse phase column and 0-100% acetonitrile (0.5% AcOH additive) in H ₂ O (0.05% AcOH additive). Elution was carried out with a mobile phase of 0.05% AcOH (additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 0.0091 g of INX N as a fluffy yellow solid in 22% yield. LCMS Method A (ESI+): C ₄₁ H ₄₈ BrN ₄ O ₁₁ S[M+H] ⁺ required 883.21 and found 883.2 in 2.247 min.

（４－（４－ホルミルベンジル）チアゾール－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－２－１）の合成：

手順：
丸底フラスコに、アルゴンを戻して充填し、（４－（ブロモメチル）チアゾール－２－イル）カルバミン酸ｔｅｒｔ－ブチル（０．１５０ｇ、０．５１１５ｍｍｏｌ、１．５当量）、（４－ホルミルフェニル）ボロン酸（０．０５１１ｇ、０．３４１１ｍｍｏｌ、１．０当量）、炭酸カリウム（０．２３５７ｇ、１．７０６ｍｍｏｌ、５．０当量）、及び［１．１′－ビス（ジフェニルホスフィノ）フェロセン］ジクロロパラジウム－ジクロロメタン複合体（０．０２７９ｇ、０．０３４１ｍｍｏｌ、０．１０当量）を充填した。無水ＴＨＦ（２．５ｍＬ）をフラスコに添加し、次いで、フラスコに還流凝縮器を装備し、８０℃まで１６時間加熱した。出発材料消費をＬＣＭＳによって確認し、次いで、混合物を冷却し、水（１０ｍＬ）で希釈し、分液漏斗に添加し、ＥｔＯＡｃ（３×２０ｍＬ）で抽出した。組み合わせた有機物をＮａ_２ＳＯ_４上で乾燥させ、濾過し、還元し、Ｉｓｃｏ Ｒｆ Ｇｏｌｄ ２４ｇ ＳｉＯ_２カラム上に装填し、ヘキサン中の０～１００％ＥｔＯＡｃの移動相で溶出した。純粋な生成物を含有する画分を組み合わせて還元し、透明な油として８％収率で０．００８２ｇの化合物ＩＮ－ＳＭ－２－１得て、これを減圧からの除去後に一晩結晶化させた。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_１６Ｈ_１９Ｎ_２Ｏ_３Ｓ［Ｍ＋Ｈ］^＋は、３１９．１０を必要とし、２．１７１６分における実測値３１９．１である。 Synthesis reaction scheme of INX-SM-2 and INX Q

Synthesis of tert-butyl (4-(4-formylbenzyl)thiazol-2-yl)carbamate (INX-SM-2-1):

procedure:
A round bottom flask was backfilled with argon and tert-butyl (4-(bromomethyl)thiazol-2-yl)carbamate (0.150 g, 0.5115 mmol, 1.5 eq.), (4-formylphenyl) Boronic acid (0.0511 g, 0.3411 mmol, 1.0 eq.), potassium carbonate (0.2357 g, 1.706 mmol, 5.0 eq.), and [1.1'-bis(diphenylphosphino)ferrocene]dichloro Palladium-dichloromethane complex (0.0279g, 0.0341mmol, 0.10eq) was charged. Anhydrous THF (2.5 mL) was added to the flask, which was then equipped with a reflux condenser and heated to 80° C. for 16 hours. Starting material consumption was confirmed by LCMS, then the mixture was cooled, diluted with water (10 mL), added to a separatory funnel, and extracted with EtOAc (3 x 20 mL). The combined organics were dried over Na ₂ SO ₄ , filtered, reduced and loaded onto an Isco Rf Gold 24g SiO ₂ column and eluted with a mobile phase of 0-100% EtOAc in hexanes. The fractions containing pure product were combined and reduced to give 0.0082 g of compound IN-SM-2-1 in 8% yield as a clear oil, which crystallized overnight after removal from vacuum. I let it happen. LCMS Method A (ESI+): _C16H19N2O3S [M+H] ⁺ requires _319.10 , _found 319.1 at 2.1716 _min .

手順：
丸底フラスコに、１６－α－ヒドロキシプレドニゾロン（０．１９３６ｇ、０．５１４３ｍｍｏｌ、１．０当量）、アルデヒドＩＮＸ－ＳＭ－２－１（０．１８００ｇ、０．５６５９ｍｍｏｌ、１．１当量）、及びＭｇＳＯ_４（０．１８５７ｇ、１．５４２８ｍｍｏｌ、３．０当量）を充填した。固体をアセトニトリル（５．１ｍＬ）中に懸濁し、混合物を０℃まで冷却すると、トリフルオロメタンスルホン酸（０．２３ｍＬ、２．５７１ｍｍｏｌ、５．０当量）を滴加した。１０～２０分後、反応物がピンクがかかった色に変わり、出発物質が約１時間後に消費された。溶媒を還元し、粗製物をＩｓｃｏ Ｃ１８ Ａｑ ３０ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として５２％収率で０．１６８０ｇのＩＮＸ－ＳＭ－２・ＡｃＯＨを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_３２Ｈ_３７Ｎ_２Ｏ_６Ｓ［Ｍ＋Ｈ］^＋は、５７７．２３を必要とし、１．９７４分における実測値５７７．３である。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((2-aminothiazol-4-yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5] Synthesis of indeno[1,2-d][1,3]dioxol-4-one AcOH salt (INX-SM-2):

procedure:
In a round bottom flask, 16-α-hydroxyprednisolone (0.1936 g, 0.5143 mmol, 1.0 eq.), aldehyde INX-SM-2-1 (0.1800 g, 0.5659 mmol, 1.1 eq.), and _MgSO4 (0.1857g, 1.5428mmol, 3.0eq) was charged. The solid was suspended in acetonitrile (5.1 mL), the mixture was cooled to 0° C., and trifluoromethanesulfonic acid (0.23 mL, 2.571 mmol, 5.0 eq.) was added dropwise. After 10-20 minutes, the reaction turned pinkish in color and the starting material was consumed after about 1 hour. The solvent was reduced and the crude was loaded onto an Isco C18 Aq 30 g reverse phase column with transfer of 0-100% acetonitrile (0.05% AcOH additive) in H ₂ O (0.05% AcOH additive). The phase eluted. Fractions containing pure product were combined, frozen, and lyophilized to yield 0.1680 g of INX-SM-2.AcOH as a white solid in 52% yield. LCMS Method A (ESI+): _C32H37N2O6S [ _M +H] ⁺ requires _577.23 , _found 577.3 at 1.974 min.

手順：
丸底フラスコに、ＩＮＸ－ＳＭ－２・ＡｃＯＨ（０．１１２５ｇ、０．１７６ｍｍｏｌ、１．０当量）、Ｂｏｃ－Ｇｌｙ－Ｇｌｕ（ＯｔＢｕ）－ＯＨ（０．０７００ｇ、０．１７６０ｍｍｏｌ、１当量）、及びＰｙＡＯＰ（０．１２２０ｇ、０．２３４０ｍｍｏｌ、１．３当量）を充填した。ＤＭＦ（１．６ｍＬ）、続いて、ＤＩＰＥＡ（０．０８１ｍＬ、０．４６８６ｍｍｏｌ、２．６当量）を添加し、混合物を室温で２時間撹拌した。ＩＮＸ－ＳＭ－２の大部分が消費されると、粗混合物をＩｓｃｏ Ｃ１８ Ａｑ １５．５ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として３７％収率で０．０６０ｇのＩＮＸ Ｑ－１を得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_４８Ｈ_６３Ｎ_４Ｏ_１２Ｓ［Ｍ＋Ｈ］^＋は、９１９．４１を必要とし、２．９３１分における実測値９１９．４である。 (S)-4-(2-((tert-butoxycarbonyl)amino)acetamide)-5-((4-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)thiazol-2-yl)amino)-5-oxopentanoic acid tert -Synthesis of butyl (INX Q-1):

procedure:
In a round bottom flask, INX-SM-2 AcOH (0.1125 g, 0.176 mmol, 1.0 equivalent), Boc-Gly-Glu(OtBu)-OH (0.0700 g, 0.1760 mmol, 1 equivalent), and PyAOP (0.1220g, 0.2340mmol, 1.3eq). DMF (1.6 mL) was added followed by DIPEA (0.081 mL, 0.4686 mmol, 2.6 eq.) and the mixture was stirred at room temperature for 2 hours. Once most of the INX-SM-2 was consumed, the crude mixture was loaded onto an Isco C18 Aq 15.5 g reverse phase column and washed with 0-100% acetonitrile in H ₂ O (0.05% AcOH additive). (0.05% AcOH additive) mobile phase. Fractions containing pure product were combined, frozen, and lyophilized to yield 0.060 g of INX Q-1 as a white solid in 37% yield. LCMS Method A (ESI+): _C48H63N4O12S [ _M +H] ⁺ requires _919.41 , _found 919.4 at 2.931 min.

手順：
丸底フラスコに、ｔｅｒｔ－ブチルエステルＩＮＸ Ｑ－１（０．０８００ｇ、０．０８７１ｍｍｏｌ、１．０当量）、ＭｅＣＮ（１．０ｍＬ）、トリフルオロ酢酸（１．０ｍＬ）、及びトリイソプロピルシラン（０．１７８ｍＬ、０．８７１ｍｍｏｌ、１０．０当量）を充填した。混合物を室温で３時間撹拌させた。出発物質の消費をＬＣＭＳによって確認し、溶媒を還元した。結果として得られた残渣を、Ｉｓｃｏ Ｃ１８ Ａｑ １５．５ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．１０％ＴＦＡ添加剤）中の０～１００％アセトニトリル（０．１０％ＴＦＡ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として１３％収率で０．０１００ｇのＩＮＸ Ｑ－２・ＴＦＡを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_３９Ｈ_４７Ｎ_４Ｏ_１０Ｓ［Ｍ＋Ｈ］^＋は、７６３．２９を必要とし、１．９４５分における実測値７６３．２である。 (S)-4-(2-aminoacetamide)-5-((4-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)thiazol-2-yl)amino)-5-oxopentanoic acid TFA salt (INX Q-2) Synthesis:

procedure:
In a round bottom flask were added tert-butyl ester INX Q-1 (0.0800 g, 0.0871 mmol, 1.0 eq), MeCN (1.0 mL), trifluoroacetic acid (1.0 mL), and triisopropylsilane (0 .178 mL, 0.871 mmol, 10.0 equivalent) was charged. The mixture was allowed to stir at room temperature for 3 hours. Consumption of starting material was confirmed by LCMS and solvent was reduced. The resulting residue was loaded onto an Isco C18 Aq 15.5 g reverse phase column and 0-100% acetonitrile (0.10% TFA additive) in H ₂ O (0.10% TFA additive). It was eluted with a mobile phase of Fractions containing pure product were combined, frozen, and lyophilized to yield 0.0100 g of INX Q-2.TFA as a white solid in 13% yield. LCMS Method A (ESI+): _C39H47N4O10S [ _M +H] ⁺ requires _763.29 , _found 763.2 at 1.945 min.

手順：
バイアルに、２－ブロモ酢酸（０．００３６ｇ、０．０２６２ｍｍｏｌ、２．３当量）を充填し、これをＤＭＦ（０．５００ｍＬ）に溶解させた。Ｎ－エトキシカルボニル－２－エトキシ－１，２－ジヒドロキノリン（０．００６２ｇ、０．０２５０ｍｍｏｌ、２．２当量）を添加し、混合物を９０分間撹拌した。次いで、アミンＩＮＸ Ｑ－２・ＴＦＡ（０．０１０ｇ、０．０１１４ｍｍｏｌ、１．０当量）を重炭酸ナトリウム（０．００６６ｇ、０．０７８６ｍｍｏｌ、６．９当量）とともに溶液に添加し、混合物を２時間（全てのＩＮＸ Ｑ－２が消費されるまで）撹拌させた。反応完了がＬＣＭＳによって確認されると、粗混合物をＩｓｃｏ Ｃ１８ Ａｑ ５．５ｇ逆相カラム上に直接装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、綿毛状の黄色の固体として３６％収率で０．００３６ｇのＩＮＸ Ｑを得た。ＬＣＭＳ方法Ｂ（ＥＳＩ＋）：Ｃ_４１Ｈ_４８ＢｒＮ_４Ｏ_１１Ｓ［Ｍ＋Ｈ］^＋は、８８３．２１を必要とし、１．２０分における実測値８８３．５３である。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)thiazol-2-yl)amino)-5-oxopentanoic acid (INX Q) Synthesis of:

procedure:
A vial was charged with 2-bromoacetic acid (0.0036g, 0.0262mmol, 2.3eq), which was dissolved in DMF (0.500mL). N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (0.0062g, 0.0250mmol, 2.2eq) was added and the mixture was stirred for 90 minutes. Amine INX Q-2 TFA (0.010 g, 0.0114 mmol, 1.0 eq.) was then added to the solution along with sodium bicarbonate (0.0066 g, 0.0786 mmol, 6.9 eq.) and the mixture was Allow to stir for an hour (until all INX Q-2 is consumed). Once reaction completion was confirmed by LCMS, the crude mixture was loaded directly onto an Isco C18 Aq 5.5 g reverse phase column and 0-100% acetonitrile (0.5% AcOH additive) in H ₂ O (0.05% AcOH additive). Elution was carried out with a mobile phase of 0.05% AcOH (additive). Fractions containing pure product were combined, frozen, and lyophilized to give 0.0036 g of INX Q as a fluffy yellow solid in 36% yield. LCMS _Method B (ESI+): _{C41H48BrN4O11S} [M+H] ⁺ requires _883.21 , _found 883.53 at 1.20 min.

３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－３－１）の合成

手順：
ｔｅｒｔ－ブチルアルコール（２０ｍＬ）中の３－（メトキシカルボニル）ビシクロ［１．１．１］ペンタン－１－カルボン酸（１０ｇ、５８．７６ｍｍｏｌ）の溶液に、ジフェニルホスホリルアジド（ＤＰＰＡ）（２０．２ｍＬ、８８．１５ｍｍｏｌ）及びトリエチルアミン（３３．０４ｍＬ、２３５．０ｍｍｏｌ）を室温で添加した。反応混合物を８０℃で１時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：１２：８８）によって精製し、白色の固体としてＩＮＸ－ＳＭ－３－１を得た（１０ｇ、７０．５５％）。^１Ｈ ＮＭＲ （ＣＤＣｌ３） δ：７．４３（ｂｓ， １Ｈ）， ３．６９（ｓ， ３Ｈ）， ２．３０（ｓ， ６Ｈ）， １．４６（ｓ， ９Ｈ）。 Synthesis reaction scheme of INX-SM-3 and INX-SM-53

Synthesis of methyl 3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentane-1-carboxylate (INX-SM-3-1)

procedure:
To a solution of 3-(methoxycarbonyl)bicyclo[1.1.1]pentane-1-carboxylic acid (10 g, 58.76 mmol) in tert-butyl alcohol (20 mL) was added diphenylphosphoryl azide (DPPA) (20.2 mL). , 88.15 mmol) and triethylamine (33.04 mL, 235.0 mmol) were added at room temperature. The reaction mixture was heated at 80°C for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane: 12:88) to obtain INX-SM-3-1 as a white solid (10 g, 70.55%). ^1H NMR (CDCl3) δ: 7.43 (bs, 1H), 3.69 (s, 3H), 2.30 (s, 6H), 1.46 (s, 9H).

手順：
ＴＨＦ：ＭｅＯＨ（３：１）（２０ｍＬ）中の３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－３－１）（５ｇ、２０．７０ｍｍｏｌ）の撹拌溶液に、ホウ化ナトリウム（３．９ｇ、１０３．５ｍｍｏｌ）を室温で添加し、更に１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を希釈ＨＣｌ水溶液でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、ＩＮＸ－ＳＭ－３－２粗生成物（４．３ｇ、９７．３８％）を生じさせた。ＬＣＭＳ：２１４．０ ［Ｍ＋Ｈ］＋；１Ｈ ＮＭＲ （ＣＤＣｌ３） δ：４．９９ （ｂｓ， １Ｈ）， ３．７２（ｓ， ２Ｈ）， １．９５（ｓ， ６Ｈ）， １．４２（ｓ， ６Ｈ）。 Synthesis of tert-butyl (3-(hydroxymethyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-2)

procedure:
Methyl 3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentane-1-carboxylate (INX-SM-3-1) (5 g) in THF:MeOH (3:1) (20 mL) , 20.70 mmol) at room temperature and stirred for an additional 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with dilute aqueous HCl and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to yield INX-SM-3-2 crude product (4.3 g, 97.38%). LCMS: 214.0 [M+H]+; 1H NMR (CDCl3) δ: 4.99 (bs, 1H), 3.72 (s, 2H), 1.95 (s, 6H), 1.42 (s, 6H).

手順：
ＤＣＭ（２ｍＬ）中の（３－（ヒドロキシメチル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３－２）（０．１ｇ、０．４６ｍｍｏｌ）の撹拌溶液に、デス・マーチンペルヨージナン（ＤＭＰ）（０．４０ｇ、４０．９３ｍｍｏｌ）を室温で添加し、３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：４０：６０）によって精製し、白色の固体としてＩＮＸ－ＳＭ－３－３を得た（０．０５０ｇ、５２％）。１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：９．５９（ｓ， １Ｈ）， ７．６８（ｂｓ， １Ｈ）， ２．１２（ｓ， ６Ｈ）， １．３７（ｓ， ９Ｈ）。 Synthesis of tert-butyl (3-formylbicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-3)

procedure:
tert-Butyl (3-(hydroxymethyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-2) (0.1 g, 0.46 mmol) in DCM (2 mL) Dess-Martin periodinane (DMP) (0.40 g, 40.93 mmol) was added to the stirred solution at room temperature and stirred for 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated NaHCO ₃ solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane: 40:60) to obtain INX-SM-3-3 as a white solid (0.050 g, 52%). 1H NMR (DMSO-d6) δ: 9.59 (s, 1H), 7.68 (bs, 1H), 2.12 (s, 6H), 1.37 (s, 9H).

手順：
ジオキサン（５ｍＬ）中の（３－ホルミルビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３－３）（０．４０ｇ、１．８９ｍｍｏｌ）の撹拌溶液に、ｐ－トルエンスルホンヒドラジド（８．８ｇ、４７．２０ｍｍｏｌ）を添加し、５０℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：３０：７０）によって精製し、白色の固体としてＩＮＸ－ＳＭ－３－４を得た（０．２８ｇ、３８．９６％）。ＬＣＭＳ：３２４．５（Ｍ－５６）；^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：１１．０７ （ｓ， １Ｈ）， ７．６６（ｄ， Ｊ＝ ８Ｈｚ， ２Ｈ）， ７．４０（ｄ， Ｊ＝８Ｈｚ， ２Ｈ）， ７．２３（ｓ， １Ｈ）， ２．３８（ｓ， ３Ｈ）， １．９０（ｓ， ６Ｈ）， １．３６（ｓ， ９Ｈ）。 Synthesis of tert-butyl (3-((2-tosylhydrazono)methyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-4)

procedure:
Stirred solution of tert-butyl (3-formylbicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-3) (0.40 g, 1.89 mmol) in dioxane (5 mL). To the mixture, p-toluenesulfone hydrazide (8.8 g, 47.20 mmol) was added and stirred at 50° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane: 30:70) to obtain INX-SM-3-4 as a white solid (0.28 g, 38.96%). LCMS: 324.5 (M-56); ¹ H NMR (DMSO-d6) δ: 11.07 (s, 1H), 7.66 (d, J = 8Hz, 2H), 7.40 (d, J =8Hz, 2H), 7.23(s, 1H), 2.38(s, 3H), 1.90(s, 6H), 1.36(s, 9H).

手順：
ジオキサン（３０ｍＬ）中のｔｅｒｔ－ブチル）－（３－（（２－トシルヒドラゾノ）メチル）ビシクロ［１．１．１］ペンタン－１－イル）カルバメート（ＩＮＸ－ＳＭ－３－４）（３．２０ｇ、８．４３ｍｍｏｌ）の撹拌溶液に、（４－ホルミルフェニル）ボロン酸（１．６４ｇ、８．４３ｍｍｏｌ）及びＫ_２ＣＯ_３（１．７４ｇ、１２．６４ｍｍｏｌ）を室温で添加し、１１０℃で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：１５：８５）によって精製し、白色の固体としてＩＮＸ－ＳＭ－３－５を得た（０．８１ｇ、３１．８７％）。ＬＣＭＳ：３０２．５（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：９．９７ （ｓ， １Ｈ）， ７．８４（ｄ， Ｊ＝ ７．６Ｈｚ， ２Ｈ）， ７．３３（ｄ， Ｊ＝ ７．６Ｈｚ， ２Ｈ）， ２．８９（ｓ， ２Ｈ）， １．６８（ｓ， ６Ｈ）， １．３３（ｓ， ９Ｈ）。 Synthesis of tert-butyl (3-(4-formylbenzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-5)

procedure:
tert-Butyl)-(3-((2-tosylhydrazono)methyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-4) (3.20 g) in dioxane (30 mL) , 8.43 mmol) at room temperature were added (4-formylphenyl)boronic acid (1.64 g, 8.43 mmol) and K ₂ CO ₃ (1.74 g, 12.64 mmol) at 110 °C The mixture was further stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane: 15:85) to obtain INX-SM-3-5 as a white solid (0.81 g, 31.87%). LCMS: 302.5 (M+H) ⁺ ; ¹ H NMR (DMSO-d6) δ: 9.97 (s, 1H), 7.84 (d, J = 7.6Hz, 2H), 7.33 (d, J = 7.6Hz, 2H), 2.89 (s, 2H), 1.68 (s, 6H), 1.33 (s, 9H).

手順：
ＤＣＭ（１０ｍＬ）中の（３－（４－ホルミルベンジル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３－５）（１．０ｇ、３．３１ｍｍｏｌ）及び（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（１．２４ｇ、３．３１ｍｍｏｌ）の溶液に、ＰＴＳＡ（０．９５ｇ、４．９７ｍｍｏｌ）を添加し、室温で更に１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、異性体の混合物として粗生成物を得た。粗製物を分取ＨＰＬＣによって精製し、次いで、異性体をキラル分取ＨＰＬＣ（カラム：ＩＧ ２５０×２１μｍ、５ミクロン、移動相：Ａ＝ヘプタン中の０．１％アンモニア、Ｂ＝ＩＰＡ：ＡＣＮ（７０：３０）、Ａ：Ｂ＝６０：４０）によって分離し、異性体１及び異性体２を得た。これらの異性体を、６．７２分（異性体１）及び１１．８７分（異性体２）の保持時間で溶出した。
ＩＮＸ－ＳＭ－３（異性体１）：ＬＣＭＳ：５６１．０（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：５．４５（ｓ， １Ｈ， アセタール－Ｈ）， ５．０７（ｄ， Ｊ＝５．２Ｈｚ， １Ｈ， Ｃ１６Ｈ）
ＩＮＸ－ＳＭ－５３（異性体２）：ＬＣＭＳ ５６１．１（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：６．１３（ｓ， １Ｈ， アセタール－Ｈ）， ５．４１（ｄ， Ｊ＝５．６Ｈｚ， １Ｈ， Ｃ１６Ｈ） (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)-7 -Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2' , 1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-3), and (6aR, 6bS, 7S, 8aS, 8bS, 10S, 11aR, 12aS,12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a -dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d Synthesis of [1,3]dioxol-4-one (INX-SM-53)

procedure:
tert-butyl (3-(4-formylbenzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-5) (1.0 g, 3.0 g in DCM (10 mL)). 31 mmol) and (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8 ,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (1.24 g, 3.31 mmol) To the solution was added PTSA (0.95 g, 4.97 mmol) and stirred for an additional 16 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product as a mixture of isomers. The crude was purified by preparative HPLC, and the isomers were then purified by chiral preparative HPLC (column: IG 250 x 21 μm, 5 microns, mobile phase: A = 0.1% ammonia in heptane, B = IPA:ACN ( 70:30) and A:B=60:40) to obtain isomer 1 and isomer 2. These isomers eluted with retention times of 6.72 minutes (isomer 1) and 11.87 minutes (isomer 2).
INX-SM-3 (isomer 1): LCMS: 561.0 (M+H) ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton assignment): δ: 5.45 (s, 1H, acetal-H), 5.07 (d, J=5.2Hz, 1H, C16H)
INX-SM-53 (isomer 2): LCMS 561.1 (M+H) ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton assignment): δ: 6.13 (s, 1H, acetal-H), 5 .41 (d, J=5.6Hz, 1H, C16H)

（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）－Ｌ－グルタミン酸１－ベンジル５－（ｔｅｒｔ－ブチル）の合成
（ＩＮＸ－Ｐ－１）

手順：
５００ｍＬの三つ口丸底フラスコに、ＤＭＦ（２００ｍＬ）中の（Ｓ）－２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（２５ｇ、５８．８２ｍｍｏｌ）及び重炭酸ナトリウム（９．８ｇ、１１６．６６ｍｍｏｌ）を充填した。この懸濁液に、臭化ベンジル（１０．９ｇ、６３．７４ｍｍｏｌ）を室温で添加し、室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層を水で洗浄し、Ｎａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をジエチルエーテル及びペンタンで粉砕し、白色の固体としてＩＮＸ Ｐ－１を得た（２６ｇ、８５．８３％）。ＬＣＭＳ：５１６．４（Ｍ＋Ｈ）^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicycle[1.1.1]pentan-1-yl)amino)-5 -Synthesis reaction scheme of oxopentanoic acid (INX-P)

Synthesis of (((9H-fluoren-9-yl)methoxy)carbonyl)-1-benzyl-5-(tert-butyl)-L-glutamate (INX-P-1)

procedure:
In a 500 mL three-neck round bottom flask, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5 in DMF (200 mL) was added. - Oxopentanoic acid (25g, 58.82mmol) and sodium bicarbonate (9.8g, 116.66mmol) were charged. Benzyl bromide (10.9 g, 63.74 mmol) was added to this suspension at room temperature and stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were washed with water, dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was triturated with diethyl ether and pentane to give INX P-1 as a white solid (26 g, 85.83%). LCMS: 516.4 (M+H) ⁺ .

手順：
５００ｍＬの一つ口丸底フラスコに、（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）－Ｌ－グルタミン酸１－ベンジル５－（ｔｅｒｔ－ブチル）（ＩＮＸ－Ｐ－１）（２６ｇ、５０．４２ｍｍｏｌ）及びＴＨＦ（２００ｍＬ）を充填した。この溶液に、ジエチルアミン（３６．８ｇ、５０４．１１ｍｍｏｌ）を添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させ、ペンタンで粉砕して、薄黄色の粘着性物質としてＩＮＸ－Ｐ－２を得た（２８ｇ）。粗生成物を、いずれの分析データもなく次のステップに直接使用した。 Synthesis of 1-benzyl 5-(tert-butyl) L-glutamate (INX-P-2)

procedure:
In a 500 mL single-necked round bottom flask, (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamic acid 1-benzyl 5-(tert-butyl) (INX-P-1) (26 g, 50 .42 mmol) and THF (200 mL). Diethylamine (36.8 g, 504.11 mmol) was added to this solution and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ , evaporated under vacuum and triturated with pentane to give INX-P-2 as a pale yellow sticky material (28 g). The crude product was used directly in the next step without any analytical data.

手順：
５００ｍＬの一つ口丸底フラスコに、（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシン（２８．０ｇ、９４．２７ｍｍｏｌ）及びＤＭＦ（２００ｍＬ）を充填した。この溶液に、ＥＤＣ．ＨＣｌ（１９．７ｇ、１０２．７６ｍｍｏｌ）、ＨＯＢＴ（１３．９ｇ、１０２．７６ｍｍｏｌ）、ＤＩＰＥＡ（２４．２ｇ、１８７．２４ｍｍｏｌ）及びＬ－グルタミン酸塩１－ベンジル５－（ｔｅｒｔ－ブチル）（ＩＮＸ－Ｐ－２）（３０．３８ｇ、１０３．２５ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物をカラムクロマトグラフィー（酢酸エチル／ヘキサン、５０：５０）によって精製し、薄黄色としてＩＮＸ－Ｐ－３を得た（１２．０ｇ、２３．６４％）。ＬＣＭＳ：５７４．４（Ｍ＋Ｈ）^＋。 Synthesis of (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-glutamic acid 1-benzyl 5-(tert-butyl) (INX-P-3)

procedure:
A 500 mL single neck round bottom flask was charged with (((9H-fluoren-9-yl)methoxy)carbonyl)glycine (28.0 g, 94.27 mmol) and DMF (200 mL). Add EDC to this solution. HCl (19.7 g, 102.76 mmol), HOBT (13.9 g, 102.76 mmol), DIPEA (24.2 g, 187.24 mmol) and L-glutamate 1-benzyl 5-(tert-butyl) (INX- P-2) (30.38 g, 103.25 mmol) was added at room temperature and stirred for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude product was purified by column chromatography (ethyl acetate/hexane, 50:50) to give INX-P-3 as a pale yellow color (12.0 g, 23.64%). LCMS: 574.4 (M+H) ⁺ .

手順：
５００ｍＬの一つ口丸底フラスコに、ＭｅＯＨ（１２０ｍＬ）中の（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシル－Ｌ－グルタミン酸塩１－ベンジル５－（ｔｅｒｔ－ブチル）（ＩＮＸ－Ｐ－３）（１２．０ｇ、２０．９５ｍｍｏｌ）を充填した。この溶液に、１０％Ｐｄ／Ｃ（２．４ｇ）を室温で添加し、水素で３～４時間パージした。ＴＬＣによって示される反応の完了後、反応混合物をセライト床を通して濾過し、濾液を真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水）によって精製して、オフホワイト色の固体としてＩＮＸ－Ｐ－４を得た（５ｇ、４９．４５％）。ＬＣＭＳ：４８３．２（Ｍ＋Ｈ）^＋。 (S)-2-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentanoic acid (INX-P-4) synthesis of

procedure:
In a 500 mL single neck round bottom flask, (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-glutamate 1-benzyl 5-(tert-butyl) (INX- P-3) (12.0 g, 20.95 mmol) was charged. To this solution was added 10% Pd/C (2.4 g) at room temperature and purged with hydrogen for 3-4 hours. After completion of the reaction as indicated by TLC, the reaction mixture was filtered through a bed of Celite and the filtrate was evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water) to give INX-P-4 as an off-white solid (5 g, 49.45%). LCMS: 483.2 (M+H) ⁺ .

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．４７ｇ、０．９７ｍｍｏｌ）、ＨＡＴＵ（０．５５ｇ、１．４５ｍｍｏｌ）、ＤＩＰＥＡ（０．２５ｇ、１．９４ｍｍｏｌ）及びＤＭＦ（４ｍＬ）を室温で充填した。この溶液に、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－３）（０．５９ｇ、１．０６ｍｍｏｌ）を室温で添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水、５０：５０）によって精製し、薄黄色の固体としてＩＮＸ－Ｐ－５を得た（０．４２ｇ、５７．３８％）。ＬＣＭＳ：１０２５．０（Ｍ＋Ｈ）^＋。 (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a , 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1. 1] Synthesis of tert-butyl pentan-1-yl)amino)-5-oxopentanoate (INX-P-5)

procedure:
In a 50 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Filled with oxopentanoic acid (INX-P-4) (0.47 g, 0.97 mmol), HATU (0.55 g, 1.45 mmol), DIPEA (0.25 g, 1.94 mmol) and DMF (4 mL) at room temperature. did. Add (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl) to this solution. phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H- Naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-3) (0.59g, 1.06mmol) was added at room temperature. The mixture was stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water, 50:50) to give INX-P-5 as a pale yellow solid (0.42 g, 57.38%). LCMS: 1025.0 (M+H) ⁺ .

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｐ－５）（０．４０ｇ、０．４１ｍｍｏｌ）及びＴＨＦ（４ｍＬ）を充填した。この溶液に、ジエチルアミン（０．４０ｇ、４．１０ｍｍｏｌ）を添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、黄色の固体としてＩＮＸ－Ｐ－６を得た（０．２３ｇ、７３．４３％）ＬＣＭＳ：８０２．１（Ｍ＋Ｈ）^＋。 (S)-4-(2-aminoacetamide)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate Synthesis of tert-butyl (INX-P-6)

procedure:
In a 50 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- tert-butyl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-P-5) (0.40 g, 0.41 mmol) and THF (4 mL) filled with. To this solution was added diethylamine (0.40 g, 4.10 mmol) and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to give INX-P-6 as a yellow solid (0.23 g, 73.43%) LCMS: 802.1 (M+H) ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸塩ｔｅｒｔ－ブチ（ＩＮＸ－Ｐ－６）（０．２３ｇ、０．２８ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、水（１ｍＬ）中のＮａ_２ＣＯ_３（０．１２ｇ、０．５７ｍｍｏｌ）溶液、続いて、ブロモアセチルブロミド（０．０２９ｇ、０．２８ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水、５０：５０）によって精製し、淡黄色の固体としてＩＮＸ－Ｐ－７を得た（０．０９０ｇ、３４．００％）。ＬＣＭＳ：９２２．９及び９２４．８（Ｍ及びＭ＋２）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5 -Synthesis of tert-butyl oxopentanoate (INX-P-7)

procedure:
In a 25 mL single neck round bottom flask, (S)-4-(2-aminoacetamide)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl ) Amino)-5-oxopentanoate tert-butyrate (INX-P-6) (0.23 g, 0.28 mmol) and DCM (2 mL) were charged. To this solution was added a solution of Na ₂ CO ₃ (0.12 g, 0.57 mmol) in water (1 mL) followed by bromoacetyl bromide (0.029 g, 0.28 mmol) at room temperature and stirred for 1 h. . After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water, 50:50) to give INX-P-7 as a pale yellow solid (0.090 g, 34.00%). LCMS: 922.9 and 924.8 (M and M+2).

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｐ－７）（０．０９０ｇ、０．０９７ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．０５５ｇ、０．４８ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を、ＩＮＸ－Ｐを取得するための分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．１％ＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ、５５：４５）、保持時間１５．５１分によって精製し、オフホワイト色の固体としてＲ異性体を得た（０．０１０ｇ、１１．８３％）。ＬＣＭＳ：８６６．８０及び８６８．８（Ｍ及びＭ＋２）；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＯＳ－ｄ６， 主要プロトン割り当て）： δ：５．４０（ｓ， １Ｈ， アセタール－Ｈ）， ４．９２（ｄ， Ｊ＝４．８ Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicycle[1.1.1]pentan-1-yl)amino)-5 -Synthesis of oxopentanoic acid (INX-P)

procedure:
In a 10 mL single neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R) , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1] tert-Butyl pentan-1-yl)amino)-5-oxopentanoate (INX-P-7) (0.090 g, 0.097 mmol) and DCM (2 mL) were charged. To this solution was added TFA (0.055 g, 0.48 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC to obtain INX-P (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.1% FA in water, B = acetonitrile, A:B, 55:45), retention time 15.51 minutes to give the R isomer as an off-white solid (0.010 g, 11.83%). LCMS: 866.80 and 868.8 (M and M+2); ¹ H NMR (400 MHz, DMOS-d6, major proton assignment): δ: 5.40 (s, 1H, acetal-H), 4.92 ( d, J=4.8 Hz, 1H, C16H).

３－（ヒドロキシメチル）ビシクロ［１．１．１］ペンタン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－４－１）の合成

手順：
ＴＨＦ（１５ｍＬ）中の３－（メトキシカルボニル）ビシクロ［１．１．１］ペンタン－１－カルボン酸（１０ｇ、５８．７５ｍｍｏｌ）の溶液に、ボランジメチルスルフィド（ＢＨ３．ＤＭＳ）（１３．４９ｍＬ、１７６．２ｍｍｏｌ）を０℃で滴加した。反応混合物を０℃で更に３０分間撹拌させた。ＴＬＣによって示される反応の完了後、希釈ＨＣｌ溶液をゆっくりと添加することによって、反応混合物をクエンチした。生成物を酢酸エチルで抽出し、組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、ゴム状の固体として表題化合物を得た（８．２ｇ、８９．３０％）。粗製物を次のステップに進めた。^１Ｈ ＮＭＲ （ＣＤＣｌ３） δ：３．６８（ｓ， ３Ｈ）， ３．６３（ｓ， ２Ｈ）， ３．０７（ｂｓ， １Ｈ）， ２．０５（ｓ， ６Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(3-(3-aminobenzyl)bicyclo[1.1.1]pentan-1-yl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' :4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-4) and (6aR, 6bS, 7S, 8aS, 8bS, 10S, 11aR, 12aS, 12bS) -10-(3-(3-aminobenzyl)bicyclo[1.1.1]pentan-1-yl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2, 6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole -4-one (INX-SM-54) synthesis reaction scheme

Synthesis of methyl 3-(hydroxymethyl)bicyclo[1.1.1]pentane-1-carboxylate (INX-SM-4-1)

procedure:
To a solution of 3-(methoxycarbonyl)bicyclo[1.1.1]pentane-1-carboxylic acid (10 g, 58.75 mmol) in THF (15 mL) was added borane dimethyl sulfide (BH3.DMS) (13.49 mL, 176.2 mmol) was added dropwise at 0°C. The reaction mixture was allowed to stir for an additional 30 minutes at 0°C. After completion of the reaction as indicated by TLC, the reaction mixture was quenched by slowly adding dilute HCl solution. The product was extracted with ethyl acetate and the combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a gummy solid (8.2 g, 89.30%) . The crude material was taken to the next step. ^1H NMR (CDCl3) δ: 3.68 (s, 3H), 3.63 (s, 2H), 3.07 (bs, 1H), 2.05 (s, 6H).

手順：
ＤＣＭ（２４０ｍＬ）中の３－（ヒドロキシメチル）ビシクロ［１．１．１］ペンタン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－４－１）（８．０ｇ、５６．２７ｍｍｏｌ）の撹拌溶液に、デス・マーチンペルヨージナン（ＤＭＰ）（２３．８７ｇ、５６．２７ｍｍｏｌ）を０℃で添加し、室温で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＮａＨＣＯ_３の飽和溶液でクエンチした。反応混合物をＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、ゴム状の白色の固体として表題化合物を得た（１２ｇ、粗製）。粗生成物を精製することなく次のステップに進めた。 Synthesis of methyl 3-formylbicyclo[1.1.1]pentane-1-carboxylate (INX-SM-4-2)

procedure:
To a stirred solution of methyl 3-(hydroxymethyl)bicyclo[1.1.1]pentane-1-carboxylate (INX-SM-4-1) (8.0 g, 56.27 mmol) in DCM (240 mL) was added Dess-Martin periodinane (DMP) (23.87 g, 56.27 mmol) was added at 0° C. and stirred for an additional 2 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with a saturated solution of _NaHCO3 . The reaction mixture was extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a gummy white solid (12 g, crude). The crude product was carried on to the next step without purification.

手順：
ジオキサン（１２０ｍＬ）中の３－ホルミルビシクロ［１．１．１］ペンタン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－４－２）（８ｇ、５１．８８ｍｍｏｌ）及びｐ－トルエンスルホニルヒドラジド（９．６６ｇ、５１．８８ｍｍｏｌ）の混合物を、５０℃で２時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：６０：４０）によって精製し、白色の固体として表題化合物を得た（１０ｇ、６０．３４％）。ＬＣＭＳ：３２３．２ （Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：１１．１９ （ｓ， １Ｈ）， ７．６６（ｄ， Ｊ＝ ８Ｈｚ， ２Ｈ）， ７．４０（ｄ， Ｊ＝８Ｈｚ， ２Ｈ）， ７．２０（ｓ， １Ｈ）， ３．６０（ｓ， ３Ｈ）， ２．３８（ｓ， ３Ｈ）， ２．０９（ｓ， ６Ｈ）。 Synthesis of methyl 3-((2-tosylhydrazono)methyl)bicyclo[1.1.1]pentane-1-carboxylate (INX-SM-4-3)

procedure:
Methyl 3-formylbicyclo[1.1.1]pentane-1-carboxylate (INX-SM-4-2) (8 g, 51.88 mmol) and p-toluenesulfonyl hydrazide (9.66 g) in dioxane (120 mL) , 51.88 mmol) was heated at 50° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 60:40) to give the title compound as a white solid (10 g, 60.34%). LCMS: 323.2 (M+H) ⁺ ; ¹ H NMR (DMSO-d6) δ: 11.19 (s, 1H), 7.66 (d, J= 8Hz, 2H), 7.40 (d, J= 8Hz, 2H), 7.20 (s, 1H), 3.60 (s, 3H), 2.38 (s, 3H), 2.09 (s, 6H).

手順：
ジオキサン（３０ｍＬ）中の３－（（２－トシルヒドラゾノ）メチル）ビシクロ［１．１．１］ペンタン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－４－３）（４ｇ、１２．４２ｍｍｏｌ）の撹拌溶液に、（４－ニトロフェニル）ボロン酸（２．０７ｇ、１２．４２ｍｍｏｌ）及びＫ_２ＣＯ_３（２．５７ｇ、１８．６３ｍｍｏｌ）を室温で添加し、１１０℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：０６：９４）によって精製し、白色の固体として表題化合物を得た（０．５２０ｇ、１６．０４％）。^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：８．１０（ｄ， Ｊ＝ ６．４Ｈｚ， １Ｈ）， ８．０１（ｓ， １Ｈ）， ７．６４－７．５９（ｍ， ２Ｈ）， ３．５５（ｓ， ３Ｈ）， ２．９５（ｓ， ２Ｈ）， １．８２（ｓ， ６Ｈ）。 Synthesis of methyl 3-(3-nitrobenzyl)bicyclo[1.1.1]pentane-1-carboxylate (INX-SM-4-4)

procedure:
Stirred solution of methyl 3-((2-tosylhydrazono)methyl)bicyclo[1.1.1]pentane-1-carboxylate (INX-SM-4-3) (4 g, 12.42 mmol) in dioxane (30 mL). To the mixture, (4-nitrophenyl)boronic acid (2.07 g, 12.42 mmol) and K ₂ CO ₃ (2.57 g, 18.63 mmol) were added at room temperature and stirred at 110° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 06:94) to give the title compound as a white solid (0.520 g, 16.04%). ^1H NMR (DMSO-d6) δ: 8.10 (d, J = 6.4Hz, 1H), 8.01 (s, 1H), 7.64-7.59 (m, 2H), 3.55 (s, 3H), 2.95 (s, 2H), 1.82 (s, 6H).

手順：
ＤＣＭ（２５ｍＬ）中の３－（３－ニトロベンジル）ビシクロ［１．１．１］ペンタン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－４－４）（０．４９０ｇ、１．８７ｍｍｏｌ）の撹拌溶液に、水素化ジイソブチルアルミニウム（トルエン中の１Ｍ、３．２ｍＬ、３．７５ｍｍｏｌ）を－７８℃で添加し、更に３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を希釈ＨＣｌ溶液でクエンチし、室温にさせ、次いで、ＤＣＭで抽出した。組み合わせた有機層をブラインで洗浄し、Ｎａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：１８：８２）によって精製し、白色の固体として表題化合物を得た（０．２７ｇ、６２．２６％）。^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：９．５５（ｓ， １Ｈ）， ８．１２（ｄ， Ｊ＝ ８Ｈｚ， １Ｈ）， ７．９９（ｓ， １Ｈ）， ７．５１（ｔ， Ｊ＝７．６ Ｈｚ， １Ｈ）， ７．４４（ｄ， Ｊ＝７．６Ｈｚ， １Ｈ）， ２．９５（ｓ， ２Ｈ）， １．９３（ｓ， ６Ｈ）。 Synthesis of 3-(3-nitrobenzyl)bicyclo[1.1.1]pentane-1-carbaldehyde (INX-SM-4-5)

procedure:
Stirred solution of methyl 3-(3-nitrobenzyl)bicyclo[1.1.1]pentane-1-carboxylate (INX-SM-4-4) (0.490 g, 1.87 mmol) in DCM (25 mL). To the solution, diisobutylaluminum hydride (1M in toluene, 3.2 mL, 3.75 mmol) was added at −78° C. and stirred for an additional 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with dilute HCl solution, allowed to reach room temperature, and then extracted with DCM. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 18:82) to give the title compound as a white solid (0.27 g, 62.26%). ^1H NMR (DMSO-d6) δ: 9.55 (s, 1H), 8.12 (d, J = 8Hz, 1H), 7.99 (s, 1H), 7.51 (t, J = 7 .6 Hz, 1H), 7.44 (d, J=7.6Hz, 1H), 2.95 (s, 2H), 1.93 (s, 6H).

手順：
ＤＣＭ（３０ｍＬ）中の３－（３－ニトロベンジル）ビシクロ［１．１．１］ペンタン－１－カルバルデヒド（ＩＮＸ－ＳＭ－４－５）（０．２７ｇ、１．１６ｍｍｏｌ）の撹拌溶液に、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．３５１ｇ、０．９３ｍｍｏｌ）及びｐ－トルエンスルホン酸（０．３０ｇ、１．７６ｍｍｏｌ）を添加した。反応混合物を室温で更に１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、異性体の混合物として表題化合物を得た（０．４７０ｇ、粗製）。ＬＣＭＳ：５９０．９３（Ｍ＋Ｈ）^＋。
更に、異性体をキラル分取ＨＰＬＣ（カラム：ＩＧ ２５０×２１μｍ、５ミクロン、移動相：Ａ＝ヘプタン中の０．１％アンモニア、Ｂ＝ＩＰＡ：ＡＣＮ（７０：３０）、Ａ：Ｂ＝７５：２５）によって分離し、異性体１及び異性体２を得た。これらの異性体を、１２．８５分（異性体１）及び１９．４０分（異性体２）の保持時間で溶出した。
異性体１：^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＣＤＣｌ３） Ｆｒ－１： δ ４．９４（ｄ， １Ｈ， Ｃ１６Ｈ）， ４．５７（ｓ， １Ｈ， アセタール－Ｈ）
異性体２：^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＣＤＣｌ３） Ｆｒ－１： δ ５．１９（ｄ， １Ｈ， Ｃ１６Ｈ）， ５．０８（ｓ， １Ｈ， アセタール－Ｈ） (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-10-(3-(3-nitrobenzyl) bicyclo[1.1.1]pentan-1-yl)-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-4-6)

procedure:
To a stirred solution of 3-(3-nitrobenzyl)bicyclo[1.1.1]pentane-1-carbaldehyde (INX-SM-4-5) (0.27 g, 1.16 mmol) in DCM (30 mL) , (8S,9S,10R,11S,13S,14S,16R,17S-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9, 10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0.351 g, 0.93 mmol) and p-toluene Sulfonic acid (0.30 g, 1.76 mmol) was added. The reaction mixture was stirred at room temperature for an additional 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with DCM. The layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a mixture of isomers (0.470 g, crude). LCMS: 590.93 (M+H) ⁺ .
The isomers were further purified by chiral preparative HPLC (column: IG 250 x 21 μm, 5 microns, mobile phase: A = 0.1% ammonia in heptane, B = IPA:ACN (70:30), A:B = 75 :25) to obtain isomer 1 and isomer 2. These isomers eluted with retention times of 12.85 minutes (isomer 1) and 19.40 minutes (isomer 2).
Isomer 1: ^1H NMR (400 MHz, CDCl3) Fr-1: δ 4.94 (d, 1H, C16H), 4.57 (s, 1H, acetal-H)
Isomer 2: ^1H NMR (400 MHz, CDCl3) Fr-1: δ 5.19 (d, 1H, C16H), 5.08 (s, 1H, acetal-H)

手順：
エタノール（１０ｍＬ）中の（ＩＮＸ－ＳＭ－４－６、異性体の混合物）（０．３０ｇ、０．５０ｍｍｏｌ）の撹拌溶液に、ＮＨ_４Ｃｌ（０．２２ｇ、４．０ｍｍｏｌ）及びＺｎ粉塵（０．２６ｇ、４．０ｍｍｏｌ）を添加した。反応混合物を８０℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を濾過し、濾液を真空下で蒸発させて、異性体の混合物として表題化合物を得た（０．３６０ｇ、粗製）。
更に、異性体をキラル分取ＨＰＬＣ（カラム：ＩＧ ２５０×２１μｍ、５ミクロン、移動相：Ａ＝ヘプタン中の０．１％アンモニア、Ｂ＝ＩＰＡ：ＡＣＮ（７０：３０）、Ａ：Ｂ＝８２：１８）によって分離し、異性体１及び異性体２を得た。これらの異性体を、２７．９６分（異性体１）及び４３．９０分（異性体２）の保持時間で溶出した。
ＩＮＸ－ＳＭ－４（異性体１）：ＬＣＭＳ：５６０．９０（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て） δ：５．００－４．９０ （ｍ， ２Ｈ， アセタール及びＣ１６－Ｈ）
ＩＮＸ－ＳＭ－５４（異性体２：ＬＣＭＳ：５６１．００（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）） δ：５．１６ （ｄ， Ｊ ＝ ７．２Ｈｚ， １Ｈ， Ｃ１６－Ｈ）， ５．０９ （ｓ， １Ｈ， アセタール－Ｈ） (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(3-(3-aminobenzyl)bicyclo[1.1.1]pentan-1-yl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' :4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-4) and (6aR, 6bS, 7S, 8aS, 8bS, 10S, 11aR, 12aS, 12bS) -10-(3-(3-aminobenzyl)bicyclo[1.1.1]pentan-1-yl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2, 6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole Synthesis of -4-one (INX-SM-54)

procedure:
To a stirred solution of (INX-SM-4-6, mixture of isomers) (0.30 g, 0.50 mmol) in ethanol (10 mL) was added NH ₄ Cl (0.22 g, 4.0 mmol) and Zn dust ( 0.26 g, 4.0 mmol) was added. The reaction mixture was stirred at 80°C for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was filtered and the filtrate was evaporated under vacuum to give the title compound as a mixture of isomers (0.360 g, crude).
The isomers were further analyzed by chiral preparative HPLC (column: IG 250 x 21 μm, 5 microns, mobile phase: A = 0.1% ammonia in heptane, B = IPA:ACN (70:30), A:B = 82 :18) to obtain isomer 1 and isomer 2. These isomers eluted with retention times of 27.96 minutes (isomer 1) and 43.90 minutes (isomer 2).
INX-SM-4 (isomer 1): LCMS: 560.90 (M+H) ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton assignment) δ: 5.00-4.90 (m, 2H, acetal and C16-H)
INX-SM-54 (isomer 2: LCMS: 561.00 (M+H) ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton assignment)) δ: 5.16 (d, J = 7.2 Hz, 1 H, C16-H), 5.09 (s, 1H, acetal-H)

（Ｓ）－４－（２－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）アセトアミド）－５－（（３－（（３－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）アミノ－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ Ｏ－１）の合成：

手順：
丸底フラスコに、ＩＮＸ－ＳＭ－４（０．０５０ｇ、０．０８９４ｍｍｏｌ、１．０当量）、Ｂｏｃ－Ｇｌｙ－Ｇｌｕ（ＯｔＢｕ）－ＯＨ（０．０８０５ｇ、０．２２３５ｍｍｏｌ、２．５当量）、及びＰｙＡＯＰ（０．１１６５ｇ、０．２２３５ｍｍｏｌ、２．５当量）を充填した。ＤＭＦ（０．１０ｍＬ）、続いてＤＩＰＥＡ（０．０７８ｍＬ、０．４４７０ｍｍｏｌ、５．０当量）を添加し、混合物を４５分間撹拌した。この時点で、全てのＩＮＸ－ＳＭ－４を消費し、所望の生成物対ビスＧｌｙ－Ｇｌｕ結合化合物の２：１比があった。粗混合物をＩｓｃｏ Ｃ１８ Ａｑ ３０ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の５～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として２８％収率で０．０２２０ｇのＩＮＸ Ｏ－１を得た。ＬＣＭＳ方法Ｂ（ＥＳＩ＋）：Ｃ_５０Ｈ_６８Ｎ_３Ｏ_１２［Ｍ＋Ｈ］^＋は、９０２．４７を必要とし、１．７６分で９０２．８８を見出した。 S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-((3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)bicyclo[1.1.1]pentan-1-yl)methyl)phenyl)amino) -5-oxopentanoic acid (INX O) synthesis reaction scheme

(S)-4-(2-((tert-butoxycarbonyl)amino)acetamide)-5-((3-((3-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS )-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)bicyclo[1.1.1]pentan-1-yl)methyl) Synthesis of tert-butyl phenyl)amino-5-oxopentanoate (INX O-1):

procedure:
In a round bottom flask, INX-SM-4 (0.050 g, 0.0894 mmol, 1.0 eq.), Boc-Gly-Glu(OtBu)-OH (0.0805 g, 0.2235 mmol, 2.5 eq.), and PyAOP (0.1165g, 0.2235mmol, 2.5eq). DMF (0.10 mL) was added followed by DIPEA (0.078 mL, 0.4470 mmol, 5.0 eq) and the mixture was stirred for 45 minutes. At this point, all the INX-SM-4 was consumed and there was a 2:1 ratio of desired product to bisGly-Glu binding compound. The crude mixture was loaded onto an Isco C18 Aq 30g reverse phase column and eluted with a mobile phase of 5-100% acetonitrile (0.05% AcOH additive) in H ₂ O (0.05% AcOH additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 0.0220 g of INX O-1 as a white solid in 28% yield. LCMS Method B (ESI+): _C50H68N3O12 [ _M +H] ⁺ required _902.47 and found 902.88 in 1.76 _min .

手順：
丸底フラスコに、ｔｅｒｔ－ブチルエステルＩＮＸ Ｏ－１（０．０２０ｇ、０．０２２ｍｍｏｌ、１．０当量）、ＭｅＣＮ（０．５０ｍＬ）、トリフルオロ酢酸（１．０ｍＬ）、及びトリイソプロピルシラン（０．０７５ｍＬ、０．３６６２ｍｍｏｌ、１６．６当量）を充填した。混合物を室温で１時間撹拌させた。出発物質の消費をＬＣＭＳによって確認し、溶媒を還元した。結果として得られた残渣を、Ｉｓｃｏ Ｃ１８ Ａｑ ３０ｇ逆相カラム上に装填し、Ｈ_２Ｏ（０．１０％ＴＦＡ添加剤）中の０～１００％アセトニトリル（０．１０％ＴＦＡ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、白色の固体として７６％収率で０．０１４４ｇのＩＮＸ Ｏ－２・ＴＦＡを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_４１Ｈ_５２Ｎ_３Ｏ_１０［Ｍ＋Ｈ］^＋は、７４６．３６を必要とし、２．０８８分で７４６．３を見出した。 (S)-4-(2-aminoacetamide)-5-((3-((3-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b- (2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2', 1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)bicyclo[1.1.1]pentan-1-yl)methyl)phenyl)amino)-5-oxo Synthesis of pentanoic acid TFA salt (INX O-2):

procedure:
In a round bottom flask, tert-butyl ester INX O-1 (0.020 g, 0.022 mmol, 1.0 eq.), MeCN (0.50 mL), trifluoroacetic acid (1.0 mL), and triisopropylsilane (0 0.075 mL, 0.3662 mmol, 16.6 eq). The mixture was allowed to stir at room temperature for 1 hour. Consumption of starting material was confirmed by LCMS and solvent was reduced. The resulting residue was loaded onto an Isco C18 Aq 30 g reverse phase column and transferred from 0 to 100% acetonitrile (0.10% TFA additive) in H ₂ O (0.10% TFA additive). The phase eluted. Fractions containing pure product were combined, frozen, and lyophilized to yield 0.0144 g of INX O-2.TFA as a white solid in 76% yield. LCMS Method A (ESI+): C ₄₁ H ₅₂ N ₃ O ₁₀ [M+H] ⁺ required 746.36 and found 746.3 in 2.088 min.

手順：
バイアルに、２－ブロモ酢酸（０．０２０５ｇ、０．１４７６ｍｍｏｌ、２当量）を充填し、これをＤＭＦ（０．４０ｍＬ）に溶解させた。Ｎ－エトキシカルボニル－２－エトキシ－１，２－ジヒドロキノリン（０．０３４７ｇ、０．１４０２ｍｍｏｌ、２当量）を添加し、混合物を９０分間撹拌させた。次いで、アミンＩＮＸ Ｏ－２・ＴＦＡ（０．０６２２ｇ、０．０７２ｍｍｏｌ、１．０当量）を重炭酸ナトリウム（０．０３７１ｇ、０．４４２８ｍｍｏｌ、６．１５当量）とともに溶液に添加し、混合物を２時間（全てのＩＮＸ Ｏ－２が消費されるまで）撹拌させた。反応完了がＬＣＭＳによって確認されると、粗混合物をＩｓｃｏ Ｃ１８ Ａｑ ５．５ｇ逆相カラム上に直接装填し、Ｈ_２Ｏ（０．０５％ＡｃＯＨ添加剤）中の０～１００％アセトニトリル（０．０５％ＡｃＯＨ添加剤）の移動相で溶出した。純粋な生成物を含有する画分を組み合わせ、凍結させ、凍結乾燥させて、綿毛状の白色の固体として２０％収率で０．０１２４ｇのＩＮＸ Ｏを得た。ＬＣＭＳ方法Ａ（ＥＳＩ＋）：Ｃ_４３Ｈ_５３ＢｒＮ_３Ｏ_１１［Ｍ＋Ｈ］^＋は、８６６．２８を必要とし、２．１７４分で８６６．３を見出した。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-((3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7 -Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H- naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)bicyclo[1.1.1]pentan-1-yl)methyl)phenyl)amino )-5-oxopentanoic acid (INX O) synthesis:

procedure:
A vial was charged with 2-bromoacetic acid (0.0205 g, 0.1476 mmol, 2 eq.), which was dissolved in DMF (0.40 mL). N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (0.0347g, 0.1402mmol, 2eq) was added and the mixture was allowed to stir for 90 minutes. Amine INX O-2.TFA (0.0622 g, 0.072 mmol, 1.0 eq.) was then added to the solution along with sodium bicarbonate (0.0371 g, 0.4428 mmol, 6.15 eq.) and the mixture was Allow to stir for an hour (until all INX O-2 is consumed). Once reaction completion was confirmed by LCMS, the crude mixture was loaded directly onto an Isco C18 Aq 5.5 g reverse phase column and 0-100% acetonitrile (0.5% AcOH additive) in H ₂ O (0.05% AcOH additive). Elution was carried out with a mobile phase of 0.05% AcOH (additive). Fractions containing pure product were combined, frozen, and lyophilized to yield 0.0124 g of INX O as a fluffy white solid in 20% yield. LCMS Method A (ESI+): C ₄₃ H ₅₃ BrN ₃ O ₁₁ [M+H] ⁺ required 866.28 and found 866.3 in 2.174 min.

２－（３－ニトロフェニル）アセトアミド（ＩＮＸ－ＳＭ－６－１）の合成

手順：
ＤＣＭ（１５ｍＬ）中の２－（３－ニトロフェニル）酢酸（０．５ｇ、２．７６ｍｍｏｌ）の溶液に、塩化オキサリル（０．７１ｍＬ、８．２８ｍｍｏｌ）を０℃で滴加した。反応混合物を室温で更に１時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物を真空下で濃縮し、ゴム状の液体を得て、これをＤＣＭに溶解させ、アンモニアガスを０℃でパージした。ＴＬＣによって示される反応の完了後、反応混合物を重炭酸ナトリウム溶液をクエンチし、生成物を酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（０．３ｇ、６０．３３％）。粗製物を次のステップに進めた。ＬＣＭＳ：１８１．１（Ｍ＋Ｈ）^＋。 Synthesis reaction scheme of INX-SM-6 and INX-SM-56

Synthesis of 2-(3-nitrophenyl)acetamide (INX-SM-6-1)

procedure:
To a solution of 2-(3-nitrophenyl)acetic acid (0.5 g, 2.76 mmol) in DCM (15 mL) was added oxalyl chloride (0.71 mL, 8.28 mmol) dropwise at 0°C. The reaction mixture was allowed to stir for an additional hour at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated under vacuum to obtain a gummy liquid, which was dissolved in DCM and purged with ammonia gas at 0°C. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with sodium bicarbonate solution and the product was extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as an off-white solid (0.3 g, 60.33%). The crude material was taken to the next step. LCMS: 181.1 (M+H) ⁺ .

手順：
ＴＨＦ（５０ｍＬ）中の２－（３－ニトロフェニル）アセトアミド（ＩＮＸ－ＳＭ－６－１）（３．０ｇ、１６．６ｍｍｏｌ）の撹拌溶液に、ローソン試薬（１３．４ｇ、３３．３３ｍｍｏｌ）を室温で添加し、反応混合物を還流温度で１４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、酢酸エチルで生成物を抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：２８：７２）によって精製し、淡黄色の固体として表題化合物を得た（３．０ｇ、９１．７６％）。ＬＣＭＳ：１９７．１（Ｍ＋Ｈ）^＋；１Ｈ ＮＭＲ （ＤＭＳＯ）：９．６２， ９．５４ （２ ｂｒｓ， ２Ｈ）， ８．２８ （ｓ， １Ｈ）， ８．１３ （ｄ， Ｊ＝ ８．０ Ｈｚ， １Ｈ）， ７．８０ （ｄ， Ｊ＝７．６ Ｈｚ， １Ｈ）， ７．６３ （ｔ， Ｊ＝８．０ Ｈｚ， １Ｈ）， ３．９６ （ｓ， ２Ｈ）。 Synthesis of 2-(3-nitrophenyl)ethanethioamide (INX-SM-6-2)

procedure:
To a stirred solution of 2-(3-nitrophenyl)acetamide (INX-SM-6-1) (3.0 g, 16.6 mmol) in THF (50 mL) was added Lawson's reagent (13.4 g, 33.33 mmol). It was added at room temperature and the reaction mixture was stirred at reflux for 14 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and the product was extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 28:72) to give the title compound as a pale yellow solid (3.0 g, 91.76%). LCMS: 197.1 (M+H) ⁺ ; 1H NMR (DMSO): 9.62, 9.54 (2 brs, 2H), 8.28 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.80 (d, J=7.6 Hz, 1H), 7.63 (t, J=8.0 Hz, 1H), 3.96 (s, 2H).

手順：
ジイソプロピルエーテル（２５ｍＬ）中のギ酸メチルエチル（０．５ｇ、６．７５ｍｍｏｌ）及び２－クロロ酢酸エチル（０．８２４ｇ、６．７５ｍｍｏｌ）の溶液に、ｔｅｒｔ－ブトキシドカリウム（０．７５ｇ、６．７５ｍｍｏｌ）を０℃で添加し、室温で３時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物をジエチルエーテルで粉砕することによって精製し、真空下で乾燥させて、黄色の固体として表題化合物を得た（０．５５ｇ、７１．４０％）。^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：８．９４ （ｓ， １Ｈ）， ３．９４ （ｑ， ２Ｈ）， １．１１ （ｔ， ３Ｈ）。 Synthesis of potassium 2-chloro-3-ethoxy-3-oxoprop-1-en-1-oleate (INX-SM-6-3)

procedure:
To a solution of methyl ethyl formate (0.5 g, 6.75 mmol) and ethyl 2-chloroacetate (0.824 g, 6.75 mmol) in diisopropyl ether (25 mL) was added potassium tert-butoxide (0.75 g, 6.75 mmol). ) was added at 0°C and allowed to stir at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude was purified by trituration with diethyl ether and dried under vacuum to give the title compound as a yellow solid (0.55g, 71.40%). ^1H NMR (DMSO-d6) δ: 8.94 (s, 1H), 3.94 (q, 2H), 1.11 (t, 3H).

手順：
カリウム２－クロロ－３－エトキシ－３－オキソプロプ－１－エン－１－オレート（ＩＮＸ－ＳＭ－６－３）（５．５ｇ）を、希釈ＨＣｌによって処理し、酢酸エチルによって抽出し、Ｎａ_２ＳＯ_４上で乾燥させ、濃縮して、２－クロロ－３－オキソプロパン酸エチルの黄色の半固体を得た（３．０ｇ）。エタノール（５０ｍＬ）の２－（３－ニトロフェニル）エタンチオアミド（ＩＮＸ－ＳＭ－６－２）（３ｇ、１５．３０ｍｍｏｌ）の撹拌溶液に、２－クロロ－３－オキソプロパン酸エチル（２．７５ｇ、１８．３６ｍｍｏｌ）及びＮａ_２ＳＯ_４（８．０３ｇ、７６．５３ｍｍｏｌ）を添加し、８０℃で１２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：３０：７０）によって精製し、黄色がかった液体として表題化合物を得た（１．６ｇ、３５．８０％）。ＬＣＭＳ：２９３．４０（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （ＣＤＣｌ３） δ：８．３４ （ｓ， １Ｈ）， ８．２２－８．１９ （ｍ， ２Ｈ）， ７．７０ （ｄ， Ｊ＝ ７．６ Ｈｚ， １Ｈ）， ７．５７ （ｔ， Ｊ＝８Ｈｚ， １Ｈ）， ４．４９ （ｓ， ２Ｈ）， ４．３２ （ｑ， ２Ｈ）， １．３１ （ｔ， ３Ｈ）。 Synthesis of ethyl 2-(3-nitrobenzyl)thiazole-5-carboxylate (INX-SM-6-4)

procedure:
Potassium 2-chloro-3-ethoxy-3-oxoprop-1-en-1-oleate (INX-SM-6-3) (5.5 g) was treated with dilute HCl, extracted with ethyl acetate and extracted with Na ₂ Dry over SO ₄ and concentrate to give a yellow semi-solid of ethyl 2-chloro-3-oxopropanoate (3.0 g). To a stirred solution of 2-(3-nitrophenyl)ethanethioamide (INX-SM-6-2) (3 g, 15.30 mmol) in ethanol (50 mL) was added ethyl 2-chloro-3-oxopropanoate (2.75 g). , 18.36 mmol) and Na ₂ SO ₄ (8.03 g, 76.53 mmol) were added and stirred at 80° C. for 12 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 30:70) to give the title compound as a yellowish liquid (1.6 g, 35.80%). LCMS: 293.40 (M+H) ⁺ ; ¹ H NMR (CDCl3) δ: 8.34 (s, 1H), 8.22-8.19 (m, 2H), 7.70 (d, J= 7. 6 Hz, 1H), 7.57 (t, J=8Hz, 1H), 4.49 (s, 2H), 4.32 (q, 2H), 1.31 (t, 3H).

手順：
ＤＣＭ（１００ｍＬ）中の２－（３－ニトロベンジル）チアゾール－５－カルボン酸エチル（ＩＮＸ－ＳＭ－６－４）（１．６ｇ、５．４ｍｍｏｌ）の撹拌溶液に、水素化ジイソブチルアルミニウム（ＤＩＢＡＬ）（トルエン中の１Ｍ、１２．０５ｍｌ、１２．０５ｍｍｏｌ）を－７８℃で添加し、－７８℃で更に２０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を希釈ＨＣｌ溶液でクエンチし、室温にさせた。生成物をＤＣＭで抽出した。組み合わせた有機層をブラインで洗浄し、Ｎａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：１０：９０）によって精製し、オフホワイト色の固体として表題化合物を得た（０．４００ｇ、２９．４４％）。ＬＣＭＳ：２４９．２９（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：１０．００ （ｓ， １Ｈ）， ８．６２ （ｓ， １Ｈ）， ８．３０（ｓ， １Ｈ）， ８．１７ （ｄ， Ｊ＝ ８．０ Ｈｚ， １Ｈ）， ７．８５ （ｄ， Ｊ＝７．６ Ｈｚ， １Ｈ）， ７．６７ （ｔ， Ｊ＝８Ｈｚ， １Ｈ）， ４．６５ （ｓ， ２Ｈ）。 Synthesis of 2-(3-nitrobenzyl)thiazole-5-carbaldehyde (INX-SM-6-5)

procedure:
To a stirred solution of ethyl 2-(3-nitrobenzyl)thiazole-5-carboxylate (INX-SM-6-4) (1.6 g, 5.4 mmol) in DCM (100 mL) was added diisobutylaluminum hydride (DIBAL). ) (1M in toluene, 12.05 ml, 12.05 mmol) was added at -78°C and stirred for a further 20 minutes at -78°C. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with dilute HCl solution and allowed to reach room temperature. The product was extracted with DCM. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 10:90) to give the title compound as an off-white solid (0.400 g, 29.44%). LCMS: 249.29 (M+H) ⁺ ; ¹ H NMR (DMSO-d6) δ: 10.00 (s, 1H), 8.62 (s, 1H), 8.30 (s, 1H), 8.17 (d, J=8.0 Hz, 1H), 7.85 (d, J=7.6 Hz, 1H), 7.67 (t, J=8Hz, 1H), 4.65 (s, 2H) .

手順：
ＤＣＭ（２０ｍＬ）中の２－（３－ニトロベンジル）チアゾール－５－カルバルデヒド（（ＩＮＸ－ＳＭ－６－５）（０．４ｇ、１．０６ｍｍｏｌ）の撹拌溶液に、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．２１１ｇ、０．８４ｍｍｏｌ）及びｐ－トルエンスルホン酸（１．０ｇ、５．３０ｍｍｏｌ）を添加し、室温で８時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を重炭酸ナトリウムでクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をフラッシュクロマトグラフィー（メタノール／ＤＣＭ：６：９４）によって精製し、ジアステレオマーの混合物（ＩＮＸ－ＳＭ－６－６）として化合物を得た。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-10-(2-(3-nitrobenzyl) thiazol-5-yl)-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1 ,2-d][1,3]dioxol-4-one (INX-SM-6-7), and (6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-7-hydroxy- -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-10-(2-(3-nitrobenzyl)thiazol-5-yl)-1,2,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-56-1 ) synthesis

procedure:
To a stirred solution of 2-(3-nitrobenzyl)thiazole-5-carbaldehyde ((INX-SM-6-5) (0.4 g, 1.06 mmol) in DCM (20 mL) was added (8S,9S,10R , 11S, 13S, 14S, 16R, 17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12, 13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0.211 g, 0.84 mmol) and p-toluenesulfonic acid (1.0 g , 5.30 mmol) and stirred at room temperature for 8 h. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with sodium bicarbonate and extracted with DCM. The combined organic layers were purified over Na ₂ SO ₄ and evaporated under vacuum. The crude was purified by flash chromatography (methanol/DCM: 6:94) to give the compound as a mixture of diastereomers (INX-SM-6-6).

Furthermore, diastereomers were separated by preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-10 μm, 12 mm, mobile phase: A = 0.05% ammonia in water, B = 20% in ACN). A-line, A:B=45:55). These isomers were separated for 13.5 min (INX-SM-6-7, isomer 1) (0.030 g, 8.8%) and 18.50 min (INX-SM-56-1, isomer 2). ) (0.040 g, 11.8%).

手順：
エタノール（２ｍＬ）中の（ＩＮＸ－ＳＭ－６－７、異性体１）（０．０３０ｇ、０．０４９ｍｍｏｌ）の撹拌溶液に、ＮＨ_４Ｃｌ（０．０２０ｇ、０．３９ｍｍｏｌ）及びＺｎ金属（０．０２５ｇ、０．３９ｍｍｏｌ）を添加した。反応混合物を８０℃で２時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を濾過し、濾液を真空下で蒸発させた。粗製物を逆相分取ＨＰＬＣ（０．０５％アンモニア－アセトニトリル）によって精製し、白色の固体として表題化合物を得た（０．００５ｇ、１７．８％）。
ＩＮＸ－ＳＭ－６（Ｒ異性体）：ＬＣＭＳ：５７７．２（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：５．８６ （ｓ， １Ｈ， アセタール－Ｈ）， ５．０２ （ｄ， Ｃ－１６Ｈ）。 ((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(2-(3-aminobenzyl)thiazol-5-yl)-7-hydroxy-8b-(2-hydroxyacetyl )-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[ Synthesis of 1,2-d][1,3]dioxol-4-one (INX-SM-6)

procedure:
To a stirred solution of (INX-SM-6-7, isomer 1) (0.030 g, 0.049 mmol) in ethanol (2 mL) was added NH ₄ Cl (0.020 g, 0.39 mmol) and Zn metal (0 .025g, 0.39mmol) was added. The reaction mixture was heated at 80°C for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was filtered and the filtrate was evaporated under vacuum. The crude material was purified by reverse phase preparative HPLC (0.05% ammonia-acetonitrile) to give the title compound as a white solid (0.005g, 17.8%).
INX-SM-6 (R isomer): LCMS: 577.2 (M+H) ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton assignment): δ: 5.86 (s, 1H, acetal-H), 5.02 (d, C-16H).

手順：
エタノール（２ｍＬ）中の（ＩＮＸ－ＳＭ－５６－１、異性体２）（０．０４０ｇ、０．０６５ｍｍｏｌ）の撹拌溶液に、ＮＨ４Ｃｌ（０．０２７ｇ、０．５２ｍｍｏｌ）及びＺｎ金属（０．０３４ｇ、０．５２ｍｍｏｌ）を添加した。反応混合物を８０℃で２時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を濾過し、濾液を真空下で蒸発させた。粗製物を逆相分取ＨＰＬＣ（０．０５％アンモニア－アセトニトリル）によって更に精製し、白色の固体として表題化合物を得た（０．０１５ｇ、３９％）；ＬＣＭＳ：５７７．１（Ｍ＋Ｈ）^＋。
ＩＮＸ－ＳＭ－５６（Ｓ異性体）：ＬＣＭＳ：５７７．２（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：６．４０（ｓ， １Ｈ， アセタール－Ｈ）， ５．３３（ｄ， Ｊ＝６．０ Ｈｚ， Ｃ－１６Ｈ） (6aR, 6bS, 7S, 8aS, 8bS, 10S, 11aR, 12aS, 12bS)-10-(2-(3-aminobenzyl)thiazol-5-yl)-7-hydroxy-8b-(2-hydroxyacetyl) -6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1 ,2-d][1,3]dioxol-4-one (INX-SM-56) synthesis

procedure:
To a stirred solution of (INX-SM-56-1, isomer 2) (0.040 g, 0.065 mmol) in ethanol (2 mL) was added NH4Cl (0.027 g, 0.52 mmol) and Zn metal (0.034 g , 0.52 mmol) was added. The reaction mixture was heated at 80°C for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was filtered and the filtrate was evaporated under vacuum. The crude material was further purified by reverse phase preparative HPLC (0.05% ammonia-acetonitrile) to give the title compound as a white solid (0.015 g, 39%); LCMS: 577.1 (M+H) ⁺ .
INX-SM-56 (S isomer): LCMS: 577.2 (M+H) ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton assignment): δ: 6.40 (s, 1H, acetal-H), 5.33 (d, J=6.0 Hz, C-16H)

（２－ブロモチアゾール－５－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－７－１）の合成

手順：
ｔ－ＢｕＯＨ（５０ｍＬ）中の２－ブロモチアゾール－５－カルボン酸（５．０ｇ、２４．０ｍｍｏｌ）の溶液に、ジフェニルホスホリルアジド（ＤＰＰＡ）（７．７４ｍＬ、３６．０ｍｍｏｌ）及びトリエチルアミン（１３．４８ｍｌ、９６．１ｍｍｏｌ）を添加し、８０℃で１２時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：１０：９０）によって精製し、褐色の固体として表題化合物を得た（２．３ｇ、３４．２８％）。ＬＣＭＳ：２７８（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６）：１０．９８ （ｓ， １Ｈ）， ７．０９ （ｓ， １Ｈ） １．４６ （ｓ， ９Ｈ）。 Synthesis reaction scheme of INX-SM-7 and INX-SM-57

Synthesis of tert-butyl (2-bromothiazol-5-yl)carbamate (INX-SM-7-1)

procedure:
To a solution of 2-bromothiazole-5-carboxylic acid (5.0 g, 24.0 mmol) in t-BuOH (50 mL) was added diphenylphosphoryl azide (DPPA) (7.74 mL, 36.0 mmol) and triethylamine (13. 48 ml, 96.1 mmol) was added and stirred at 80° C. for 12 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 10:90) to give the title compound as a brown solid (2.3 g, 34.28%). LCMS: 278 (M+H) ⁺ ; ¹ H NMR (DMSO-d6): 10.98 (s, 1H), 7.09 (s, 1H) 1.46 (s, 9H).

手順：
（２－ブロモチアゾール－５－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－７－１）（１．５ｇ、５．３７ｍｍｏｌ）、ジオキサン（５０ｍＬ）の撹拌溶液に、トリブチル（ビニル）スズ（１．７０ｇ、５．３７ｍｍｏｌ）を室温で添加し、Ｎ_２（ｇ）で１５分間脱気した。テトラキストリフェニルホスフィンパラジウム（０）（０．３１０ｇ、０．２６ｍｍｏｌ）を反応混合物に添加し、反応混合物を１００℃で１２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をセライト床を通して濾過し、濾液を真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：２０：８０）によって精製し、表題化合物を得た（０．９ｇ、７４．２％）。ＬＣＭＳ：２２７．０（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６）：１０．７２（ｓ， １Ｈ）， ７．２６（ｓ， １Ｈ）， ６．７６（ｄｄ， Ｊ＝１１．２ ＆ １７．６ Ｈｚ，１Ｈ）， ５．８２（ｄ， Ｊ＝１７．６Ｈｚ， １Ｈ）， ５．４２（ｄ， Ｊ＝１１．２Ｈｚ， １Ｈ）， １．４６（ｓ， ９Ｈ）。 Synthesis of tert-butyl (2-vinylthiazol-5-yl)carbamate (INX-SM-7-2)

procedure:
To a stirred solution of tert-butyl (2-bromothiazol-5-yl)carbamate (INX-SM-7-1) (1.5 g, 5.37 mmol) and dioxane (50 mL) was added .70 g, 5.37 mmol) was added at room temperature and degassed with N2 _(g) for 15 min. Tetrakistriphenylphosphine palladium(0) (0.310 g, 0.26 mmol) was added to the reaction mixture and the reaction mixture was stirred at 100° C. for 12 hours. After completion of the reaction as indicated by TLC, the reaction mixture was filtered through a bed of Celite and the filtrate was evaporated under vacuum. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane: 20:80) to obtain the title compound (0.9 g, 74.2%). LCMS: 227.0 (M+H) ⁺ ; ¹ H NMR (DMSO-d6): 10.72 (s, 1H), 7.26 (s, 1H), 6.76 (dd, J=11.2 & 17 .6 Hz, 1H), 5.82 (d, J=17.6Hz, 1H), 5.42 (d, J=11.2Hz, 1H), 1.46 (s, 9H).

手順：
ジオキサン（５０ｍＬ）中の（２－ビニルチアゾール－５－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－７－２）（３．８ｇ、１６．８ｍｍｏｌ）の溶液に、水（２ｍｌ）中のＫ_２ＯｓＯ_４．２Ｈ_２Ｏ（０．１７９ｇ、０．４８ｍｍｏｌ）の溶液を添加した。ＮａＩＯ_４（１８．１５ｇ、８５．２ｍｍｏｌ）を水（１０ｍｌ）に溶解させ、反応混合物に添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をセライト床を通して濾過し、濾液を真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル：ヘキサン：１５：８５）によって精製し、淡黄色の固体として表題化合物を得た（２．５ｇ、６５．２２％）。ＬＣＭＳ：２２９．０（Ｍ＋Ｈ）^＋。 Synthesis of tert-butyl (2-formylthiazol-5-yl)carbamate (INX-SM-7-3)

procedure:
To a solution of tert-butyl (2-vinylthiazol-5-yl)carbamate (INX-SM-7-2) (3.8 g, 16.8 mmol) in dioxane (50 mL) was added K in water (2 mL). ₂ OsO ₄ . A solution of _2H2O (0.179g, 0.48mmol) was added. NaIO ₄ (18.15 g, 85.2 mmol) was dissolved in water (10 ml) and added to the reaction mixture and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was filtered through a bed of Celite and the filtrate was evaporated under vacuum. The crude product was purified by silica gel column chromatography (ethyl acetate: hexane: 15:85) to give the title compound as a pale yellow solid (2.5 g, 65.22%). LCMS: 229.0 (M+H) ⁺ .

手順：
ジオキサン（５０ｍＬ）中の（２－ホルミルチアゾール－５－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－７－３）（２．５ｇ、１０．９ｍｍｏｌ）の溶液に、ｐ－トルエンスルホニルヒドラジド（２．２３ｇ、１２．０ｍｍｏｌ）を添加し、反応混合物を９０℃で５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル：ヘキサン：２５：７５）によって精製し、淡黄色の固体として表題化合物を得た（２．８ｇ、６４．４８％）。ＬＣＭＳ：３９７．０（Ｍ＋Ｈ）^＋。 Synthesis of tert-butyl (2-((2-tosylhydrazono)methyl)thiazol-5-yl)carbamate (INX-SM-7-4)

procedure:
To a solution of tert-butyl (2-formylthiazol-5-yl)carbamate (INX-SM-7-3) (2.5 g, 10.9 mmol) in dioxane (50 mL) was added p-toluenesulfonyl hydrazide (2 .23 g, 12.0 mmol) was added and the reaction mixture was stirred at 90° C. for 5 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was purified by silica gel column chromatography (ethyl acetate: hexane: 25:75) to give the title compound as a pale yellow solid (2.8 g, 64.48%). LCMS: 397.0 (M+H) ⁺ .

手順：
ジオキサン（５０ｍＬ）中の（２－（（２－トシルヒドラゾノ）メチル）チアゾール－５－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－７－４）（２．８ｇ、７．０６ｍｍｏｌ）の撹拌溶液に、（４－ホルミルフェニル）ボロン酸（１．１６ｇ、７．７６ｍｍｏｌ）及びＫ_２ＣＯ_３（１．９４ｇ、１４．１２ｍｍｏｌ）を添加し、１１０℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：２０：８０）によって精製し、淡黄色の固体として表題化合物を得た（０．４ｇ、１７．７９％）。ＬＣＭＳ：３１９．０（Ｍ＋Ｈ）^＋。 Synthesis of tert-butyl (2-(4-formylbenzyl)thiazol-5-yl)carbamate (INX-SM-7-5)

procedure:
To a stirred solution of tert-butyl (2-((2-tosylhydrazono)methyl)thiazol-5-yl)carbamate (INX-SM-7-4) (2.8 g, 7.06 mmol) in dioxane (50 mL) , (4-formylphenyl)boronic acid (1.16 g, 7.76 mmol) and K ₂ CO ₃ (1.94 g, 14.12 mmol) were added and stirred at 110° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 20:80) to give the title compound as a pale yellow solid (0.4 g, 17.79%). LCMS: 319.0 (M+H) ⁺ .

手順：
ＤＣＭ（５０ｍＬ）中の（２－（４－ホルミルベンジル）チアゾール－５－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－７－５）（０．１ｇ、０．３１ｍｍｏｌ）及び（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドロニソロン）（０．１１８ｇ、０．３１ｍｍｏｌ）の撹拌溶液に、アセトリル（６．２ｍｌ）中のトリフル酸（０．１５ｇ、１．０３ｍｍｏｌ）の溶液を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＯＨ溶液に注ぎ、ＭＤＣで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、異性体の混合物として表題化合物を得た（０．０６０ｇ、粗製）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((5-aminothiazol-2-yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5] Indeno[1,2-d][1,3]dioxol-4-one (INX-SM-7) and (6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-( 4-((5-aminothiazol-2-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a ,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM- 57) synthesis

procedure:
tert-butyl (2-(4-formylbenzyl)thiazol-5-yl)carbamate (INX-SM-7-5) (0.1 g, 0.31 mmol) and (8S, 9S, 10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12 , 13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxypredronisolone) (0.118 g, 0.31 mmol) was added with acetyl ( A solution of triflic acid (0.15 g, 1.03 mmol) in 6.2 ml) was added and stirred for 1 hour at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was poured into saturated NaOH solution and extracted with MDC. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a mixture of isomers (0.060 g, crude).

Furthermore, the diastereomers were separated by preparative HPLC (column: Xbridge prep, C18, OBD 19 x 250 mm, 5 microns, mobile phase: A = 0.05% ammonia in water, B = ACN (67:33), A:B = 67:33) to give isomer 1 and isomer 2. These isomers eluted with retention times of 17.70 minutes (isomer 1) and 20.87 minutes (isomer 2).
INX-SM-7 (isomer 1, R isomer): (Yield: 0.010 g, 3%). LCMS: 577.4 (M+H) ⁺ ; ^1H NMR (400 MHz, MeOD, major proton assignment): δ: 5.47 (s, 1H, acetal-H), 5.06 (d, J=5.2Hz , 1H, C16H)
INX-SM-57 (isomer 2, S isomer): (Yield: 0.003 g, 0.6%). LCMS 577.4 (M+H) ⁺ ; ^1H NMR (400 MHz, MeOD, major proton assignment): δ: 6.03 (s, 1H, acetal-H), 5.41 (d, J=5.2Hz, 1H, C16H)

（６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－６ｂ－フルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン
（ＩＮＸ－ＳＭ－１３）、及び
（６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｓ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－６ｂ－フルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－６３）の合成
手順：
ＤＣＭ（２ｍＬ）中の（３－（４－ホルミルベンジル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３－５）（０．１８０ｇ、０．５９７ｍｍｏｌ）及び（８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－９－フルオロ－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（トリアムシノロン）（０．２５９ｇ、０．６５６ｍｍｏｌ）の溶液に、ｐ－トレンスルホン酸（０．９０８ｇ、４．７７ｍｍｏｌ）を添加し、室温で更に１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、異性体の混合物として粗生成化合物を得た。更に、粗生成物を精製し、異性体を逆相分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－１０μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％アンモニア、Ｂ＝ＡＣＮ：ＭｅＯＨ（５０：５０）によって分離した。これらの異性体を、１４分（異性体１）及び１９．５分（異性体２）の保持時間で溶出した。
ＩＮＸ－ＳＭ－１３（異性体１）：（収率：０．０３８ｇ、１１．０８％）。ＬＣＭＳ：５７８．２０（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：５．４７（ｓ， １Ｈ， アセタール－Ｈ）， ５．０５（ｄ， Ｊ＝５．２Ｈｚ， １Ｈ， Ｃ１６Ｈ）
ＩＮＸ－ＳＭ－６３（異性体２）：（収率：０．００５ｇ、１．４５％）。ＬＣＭＳ ５７８．３０（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：６．１３（ｓ， １Ｈ， アセタール－Ｈ）， ５．４２（ｄ， Ｊ＝６．８Ｈｚ， １Ｈ， Ｃ１６Ｈ） Synthesis reaction scheme of INX-SM-13 and INX-SM-63

(6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)-6b -Fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H- naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-13), and (6aS, 6bR, 7S, 8aS, 8bS, 10S, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)-6b-fluoro-7-hydroxy-8b-(2 -hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5 ] Synthesis procedure of indeno[1,2-d][1,3]dioxol-4-one (INX-SM-63):
tert-butyl (3-(4-formylbenzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-5) (0.180 g, 0.05 mL) in DCM (2 mL). 597 mmol) and (8S,9R,10S,11S,13S,14S,16R,17S)-9-fluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6 ,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (triamcinolone) (0.259 g, 0.656 mmol) solution To this, p-trensulfonic acid (0.908 g, 4.77 mmol) was added and the mixture was further stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into saturated NaHCO ₃ solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product compound as a mixture of isomers. Further, the crude product was purified and the isomers were separated by reverse phase preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-10 μm, 12 nm, mobile phase: A = 0.05% ammonia in water , B=ACN:MeOH (50:50). The isomers were eluted with retention times of 14 minutes (isomer 1) and 19.5 minutes (isomer 2).
INX-SM-13 (isomer 1): (Yield: 0.038 g, 11.08%). LCMS: 578.20 (M+H) ⁺ ; ^1H NMR (400 MHz, MeOD, major proton assignment): δ: 5.47 (s, 1H, acetal-H), 5.05 (d, J=5.2Hz , 1H, C16H)
INX-SM-63 (isomer 2): (Yield: 0.005 g, 1.45%). LCMS 578.30 (M+H) ⁺ ; ^1H NMR (400 MHz, MeOD, major proton assignment): δ: 6.13 (s, 1H, acetal-H), 5.42 (d, J=6.8Hz, 1H, C16H)

（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－２４）、及び
（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｓ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－７４）の合成
手順：
ＤＣＭ（１０ｍＬ）中の（３－（４－ホルミルベンジル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３－５）（０．５００ｇ、１．６６ｍｍｏｌ）及び（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ，１０，１０－テトラメチル１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（フロシノロンアセトニド）（０．７１６ｇ、１．６５ｍｍｏｌ）の溶液に、ｐ－トルエンスルホン酸（２．５ｇ、１３．２６ｍｍｏｌ）を添加し、室温で更に１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、異性体の混合物として粗生成物を得た。更に、粗生成物を精製し、異性体を逆相分取ＨＰＬＣ（カラム：Ｕｎｉｓｉｌ １０－１２０ Ｃ１８ Ｕｌｔｒａ、２５０×２１．２ｍｍ×１０μｍ、移動相：Ａ＝水中の０．０５％アンモニア、Ｂ＝アセトニトリル）によって分離し、異性体１及び異性体２を得た。これらの異性体を、１３．５分（異性体１）及び１９．５分（異性体２）の保持時間で溶出した。
ＩＮＸ－ＳＭ－２４（Ｒ異性体）：（収率０．１００ｇ、１０．１１％）。）。ＬＣＭＳ：５９６．２０（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：５．４８（ｓ， １Ｈ， アセタール－Ｈ）， ５．０６（ｄ， Ｊ＝４．４Ｈｚ， １Ｈ， Ｃ１６Ｈ）
ＩＮＸ－ＳＭ－７４（Ｓ異性体）：（収率０．０２０ｇ、２．０２％）。ＬＣＭＳ：５９６．２０（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：６．１７（ｓ， １Ｈ， アセタール－Ｈ）， ５．４３（ｄ， Ｊ＝７．２Ｈｚ， １Ｈ， Ｃ１６Ｈ） Synthesis reaction scheme of INX-SM-24 and INX-SM-74

(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl) -2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl 1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-24), and (2S,6aS,6bR,7S , 8aS, 8bS, 10S, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)-2,6b-difluoro-7 -Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2' , 1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-74) synthesis procedure:
tert-Butyl (3-(4-formylbenzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-5) (0.500 g, 1. 66 mmol) and (2S, 6aS, 6bR, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10,10- Tetramethyl 1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d] To a solution of [1,3]dioxol-4-one (furocinolone acetonide) (0.716 g, 1.65 mmol) was added p-toluenesulfonic acid (2.5 g, 13.26 mmol) and the mixture was heated at room temperature. The mixture was further stirred for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into saturated NaHCO ₃ solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product as a mixture of isomers. The crude product was further purified and the isomers were separated by reverse phase preparative HPLC (column: Unisil 10-120 C18 Ultra, 250 x 21.2 mm x 10 μm, mobile phase: A = 0.05% ammonia in water, B = (acetonitrile) to give isomer 1 and isomer 2. These isomers eluted with retention times of 13.5 minutes (isomer 1) and 19.5 minutes (isomer 2).
INX-SM-24 (R isomer): (Yield 0.100 g, 10.11%). ). LCMS: 596.20 (M+H) ⁺ ; ^1H NMR (400 MHz, MeOD, major proton assignment): δ: 5.48 (s, 1H, acetal-H), 5.06 (d, J=4.4Hz , 1H, C16H)
INX-SM-74 (S isomer): (Yield 0.020 g, 2.02%). LCMS: 596.20 (M+H) ⁺ ; ^1H NMR (400 MHz, MeOD, major proton assignment): δ: 6.17 (s, 1H, acetal-H), 5.43 (d, J=7.2Hz , 1H, C16H)

４－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）キュバン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－９－１）の合成

手順：
１００ｍＬの一つ口丸底フラスコに、４－メトキシカルボニルキュバンカルボン酸（２ｇ、９．６９ｍｍｏｌ）及びｔｅｒｔ－ブチルアルコール（６０ｍＬ）を充填した。この溶液に、ジフェニルホスホリルアジド（ＤＰＰＡ）（３．１ｍＬ、１４．５４ｍｍｏｌ）及びトリエチルアミン（１０．８ｍＬ、７７．５９ｍｍｏｌ）を室温で添加し、室温で３０分間撹拌した。反応混合物を８０℃で１時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：１５：８５）によって精製し、白色の固体として表題化合物を得た（０．９０ｇ、３３．４６％）。^１Ｈ ＮＭＲ （ＣＤＣｌ３） δ：４．１（ｂｓ， ６Ｈ）， ３．７１（ｓ， ３Ｈ）， １．４６（ｓ， ９Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((4-aminocuban-1-yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5] Synthesis reaction scheme of indeno[1,2-d][1,3]dioxol-4-one (INX-SM-9)

Synthesis of methyl 4-((tert-butoxycarbonyl)amino)cubane-1-carboxylate (INX-SM-9-1)

procedure:
A 100 mL single neck round bottom flask was charged with 4-methoxycarbonylcubanecarboxylic acid (2 g, 9.69 mmol) and tert-butyl alcohol (60 mL). Diphenylphosphoryl azide (DPPA) (3.1 mL, 14.54 mmol) and triethylamine (10.8 mL, 77.59 mmol) were added to this solution at room temperature and stirred at room temperature for 30 minutes. The reaction mixture was heated at 80°C for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 15:85) to give the title compound as a white solid (0.90 g, 33.46%). ^1H NMR (CDCl3) δ: 4.1 (bs, 6H), 3.71 (s, 3H), 1.46 (s, 9H).

手順：
１００ｍＬの三つ口丸底フラスコに、窒素下で４－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）キュバン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－９－１）（０．９ｇ、３．２４ｍｍｏｌ）及びＴＨＦ（４０ｍＬ）を充填した。この溶液に、ＴＨＦ中の１Ｍ水素化アルミニウムリチウム（３．２ｍＬ、３．２４ｍｍｏｌ）を－７８℃で添加し、更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を１ＮのＮａＯＨ溶液でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た（０．８ｇ、９８．８８％）。^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：７．５８（ｂｓ， １Ｈ）， ４．４２（ｔ， １Ｈ）， ３．８０（ｂｓ， ３Ｈ）， ３．５７（ｂｓ， ３Ｈ） ３．４８（ｄ， ２Ｈ， Ｊ＝５．２）， １．３７（ｓ， ９Ｈ）。 Synthesis of tert-butyl (4-(hydroxymethyl)cuban-1-yl)carbamate (INX-SM-9-2)

procedure:
In a 100 mL three-neck round bottom flask under nitrogen, methyl 4-((tert-butoxycarbonyl)amino)cubane-1-carboxylate (INX-SM-9-1) (0.9 g, 3.24 mmol) and Charged with THF (40 mL). To this solution was added 1M lithium aluminum hydride in THF (3.2 mL, 3.24 mmol) at −78° C. and stirred for an additional hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with 1N NaOH solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product (0.8 g, 98.88%). ¹ H NMR (DMSO-d6) δ: 7.58 (bs, 1H), 4.42 (t, 1H), 3.80 (bs, 3H), 3.57 (bs, 3H) 3.48 (d , 2H, J=5.2), 1.37(s, 9H).

手順：
１００ｍＬの三つ口丸底フラスコに、窒素下で（４－（ヒドロキシメチル）キュバン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－９－２）（０．９ｇ、３．６０ｍｍｏｌ）及びＤＣＭ（２５ｍＬ）を充填した。この溶液に、デス・マーチンペルヨージナン（ＤＭＰ）（３．０６ｇ、７．２１ｍｍｏｌ）を０℃で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をセライトを通して濾過し、ジエチルエーテルで洗浄した。組み合わせた濾液を真空下で蒸発させ、白色の固体として表題化合物を得た（１．０ｇ、粗製物、定量的）。粗製物を即時に次のステップに使用した。 Synthesis of 4tert-butyl (4-formylcuban-1-yl)carbamate (INX-SM-9-3)

procedure:
In a 100 mL three-neck round bottom flask under nitrogen, tert-butyl (4-(hydroxymethyl)cuban-1-yl)carbamate (INX-SM-9-2) (0.9 g, 3.60 mmol) and DCM (25 mL) was charged. Dess-Martin periodinane (DMP) (3.06 g, 7.21 mmol) was added to this solution at 0° C. and stirred for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was filtered through Celite and washed with diethyl ether. The combined filtrates were evaporated under vacuum to give the title compound as a white solid (1.0 g, crude, quantitative). The crude material was used immediately in the next step.

手順：
５０ｍＬの一つ口丸底フラスコに、窒素下で（４－ホルミルキュバン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－９－３）（１．０ｇ、４．０４ｍｍｏｌ）及びＥｔＯＨ（３０ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホニルヒドラジド（１．１ｇ、６．０６ｍｍｏｌ）を触媒量のＡｃＯＨとともに添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注いだ。固体を濾過し、生成物を真空下で乾燥させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル：ヘキサン、１：４）によって精製し、白色の固体として表題化合物を得た（０．８ｇ、４７．６１％）。ＬＣＭＳ：４１６．３（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：１１．０７（ｓ， １Ｈ）， ７．６７（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ７．４０－７．３８（ｍ， ３Ｈ）， ３．８５－３．８１（ｍ， ６Ｈ）， ２．３８（ｓ， ３Ｈ）， １．３６（ｓ， ９Ｈ）。 Synthesis of tert-butyl (4-((2-tosylhydrazono)methyl)cuban-1-yl)carbamate (INX-SM-9-4)

procedure:
In a 50 mL single neck round bottom flask, tert-butyl (4-formylcuban-1-yl)carbamate (INX-SM-9-3) (1.0 g, 4.04 mmol) and EtOH (30 mL) were added under nitrogen. ) was filled. To this solution was added p-toluenesulfonyl hydrazide (1.1 g, 6.06 mmol) along with a catalytic amount of AcOH and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water. The solids were filtered and the product was dried under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate:hexane, 1:4) to give the title compound as a white solid (0.8 g, 47.61%). LCMS: 416.3 (M+H) ⁺ ; ¹ H NMR (DMSO-d6) δ: 11.07 (s, 1H), 7.67 (d, J=8.4Hz, 2H), 7.40-7. 38 (m, 3H), 3.85-3.81 (m, 6H), 2.38 (s, 3H), 1.36 (s, 9H).

手順：
３５ｍＬのバイアルに、窒素下で（４－（（２－トシルヒドラゾノ）メチル）キュバン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－９－４）（０．５０ｇ、１．２０ｍｍｏｌ）及びジオキサン（１０ｍＬ）を充填した。反応混合物をＮ_２で１０分間パージした。この溶液に、（４－ホルミルフェニル）ボロン酸（０．３６ｇ、２．４０ｍｍｏｌ）及びＫ_２ＣＯ_３（０．３３ｇ、２．４１ｍｍｏｌ）を室温で添加し、１１０℃で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、１５：８５）によって精製し、白色の固体として表題化合物を得た（０．０４０ｇ、９．８５％）。^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：９．９６ （ｓ， １Ｈ）， ７．８３（ｄ， Ｊ＝ ８Ｈｚ， ２Ｈ）， ７．５９（ｂｓ， １Ｈ）， ７．４０（ｄ， Ｊ＝ ７．６Ｈｚ， ２Ｈ）， ３．７６（ｂｓ， ３Ｈ）， ３．５８（ｂｓ， ３Ｈ）， ２．９６（ｓ， ２Ｈ）， １．３５（ｓ， ９Ｈ）。 Synthesis of tert-butyl (4-(4-formylbenzyl)cuban-1-yl)carbamate (INX-SM-9-5)

procedure:
In a 35 mL vial, tert-butyl (4-((2-tosylhydrazono)methyl)cuban-1-yl)carbamate (INX-SM-9-4) (0.50 g, 1.20 mmol) and dioxane were added under nitrogen. (10 mL). The reaction mixture was purged with _N2 for 10 minutes. To this solution was added (4-formylphenyl)boronic acid (0.36 g, 2.40 mmol) and K ₂ CO ₃ (0.33 g, 2.41 mmol) at room temperature and stirred at 110° C. for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 15:85) to give the title compound as a white solid (0.040 g, 9.85%). ^1H NMR (DMSO-d6) δ: 9.96 (s, 1H), 7.83 (d, J= 8Hz, 2H), 7.59 (bs, 1H), 7.40 (d, J= 7 .6Hz, 2H), 3.76 (bs, 3H), 3.58 (bs, 3H), 2.96 (s, 2H), 1.35 (s, 9H).

手順：
１０ｍＬの一つ口丸底フラスコに、（（２ｒ，３Ｒ，４ｓ，５Ｓ）－４－（４－ホルミルベンジル）キュバン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－９－５）（０．０３５ｇ、０．１０ｍｍｏｌ）及び（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－アルファ－ヒドロキシプレドニゾロン）（０．０３８ｇ、０．１０ｍｍｏｌ）、ＭｇＳＯ_４（０．０６２ｇ、０．５１ｍｍｏｌ）、ならびにＤＣＭ（１０ｍＬ）を充填した。この溶液に、ＨＣｌＯ_４（０．１５７ｇ、１．５５ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、真空下で濃縮した。粗製物を冷水で粉砕し、参加したものを濾過し、真空下で乾燥させた。粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．１％ＦＡ、Ｂ＝ＡＣＮ：ＭｅＯＨ：ＩＰＡ（６５：２５：１０）、Ａ：Ｂ、６７：３３）、保持時間１５．１４分によって精製し、白色の固体としてＲ異性体を得た（０．０１０ｇ、１６．１８％）；ＬＣＭＳ：５９７．４（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：５．４７（ｓ， １Ｈ， アセタール－Ｈ）， ５．０６（ｄ， Ｊ＝４．８ Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((4-aminocuban-1-yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5] Synthesis of indeno[1,2-d][1,3]dioxol-4-one (INX-SM-9)

procedure:
In a 10 mL single-neck round bottom flask, add tert-butyl ((2r,3R,4s,5S)-4-(4-formylbenzyl)cuban-1-yl)carbamate (INX-SM-9-5) ( 0.035g, 0.10mmol) and (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl -6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-alpha-hydroxyprednisolone) (0.038g , 0.10 mmol), MgSO ₄ (0.062 g, 0.51 mmol), and DCM (10 mL). To this solution was added HClO ₄ (0.157 g, 1.55 mmol) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated _NaHCO3 solution and concentrated under vacuum. The crude was triturated with cold water and the residue was filtered and dried under vacuum. The crude product was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.1% FA in water, B = ACN:MeOH:IPA (65:25:10), A: LCMS: 597.4 (M+H) ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton assignment): δ: 5.47 (s, 1H, acetal-H), 5.06 (d, J=4.8 Hz, 1H, C16H).

（６－（ヒドロキシメチル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３２－１）の合成

手順：
５０ｍＬの一つ口丸底フラスコに、６－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）スピロ［３．３］ヘプタン－２－カルボン酸メチル（２．０ｇ、７．４３ｍｍｏｌ）及びＴＨＦ：ＭｅＯＨ（１５：５ｍＬ）を窒素下で充填した。この溶液に、ＮａＢＨ_４（１．４ｇ、３７．１７ｍｍｏｌ）を０℃で分割して添加し、室温で更に４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水で希釈し、１ＮのＨＣｌで中性ｐＨを調整した。生成物を酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た（２．０ｇ、定量的）。ＬＣＭＳ：１８６．２ （Ｍ＋Ｈ －５６）， ^１Ｈ ＮＭＲ （ＣＤＣｌ_３） δ：４．６３（ｂｓ， １Ｈ）， ３．９７（ｂｓ， １Ｈ）， ３．５４（ｄ， Ｊ＝６．８ Ｈｚ， ２Ｈ）， ２．５０－２．２５（ｍ， ３Ｈ）， ２．２０－１．９５（ｍ， ２Ｈ）， １．９０－１．４０（ｍ， ５Ｈ）， １．４６（ｓ， ９Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)methyl)phenyl)-7-hydroxy-8b- (2-Hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4 ,5] Synthesis reaction scheme of indeno[1,2-d][1,3]dioxol-4-one (INX-SM-32)

Synthesis of tert-butyl (6-(hydroxymethyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-32-1)

procedure:
In a 50 mL single neck round bottom flask, methyl 6-((tert-butoxycarbonyl)amino)spiro[3.3]heptane-2-carboxylate (2.0 g, 7.43 mmol) and THF:MeOH (15: 5 mL) under nitrogen. To this solution was added NaBH ₄ (1.4 g, 37.17 mmol) in portions at 0° C. and stirred for an additional 4 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with water and adjusted to neutral pH with 1N HCl. The product was extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product (2.0 g, quantitative). LCMS: 186.2 (M+H -56), ^1H NMR ( _CDCl3 ) δ: 4.63 (bs, 1H), 3.97 (bs, 1H), 3.54 (d, J=6.8 Hz , 2H), 2.50-2.25 (m, 3H), 2.20-1.95 (m, 2H), 1.90-1.40 (m, 5H), 1.46 (s, 9H) ).

手順：
５０ｍＬの一つ口丸底フラスコに、窒素下で（６－（ヒドロキシメチル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３２－１）（２．０ｇ、８．３０ｍｍｏｌ）及びＤＣＭ（２０ｍＬ）を充填した。この溶液に、デス・マーチンペルヨージナン（ＤＭＰ）（３．５１ｇ、８．３０ｍｍｏｌ）を０℃で添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をセライトを通して濾過し、ジエチルエーテルで洗浄した。組み合わせた有機層を真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、４０：６０）によって精製し、黄色の固体の／として表題化合物を得た（１．７ｇ、８５．７２％）。ＬＣＭＳ：１８４．２ （Ｍ＋Ｈ－５６）。 Synthesis of tert-butyl (6-formylspiro[3.3]heptan-2-yl)carbamate (INX-SM-32-2)

procedure:
In a 50 mL single neck round bottom flask under nitrogen, add tert-butyl (6-(hydroxymethyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-32-1) (2.0 g). , 8.30 mmol) and DCM (20 mL). Dess-Martin periodinane (DMP) (3.51 g, 8.30 mmol) was added to this solution at 0° C. and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was filtered through Celite and washed with diethyl ether. The combined organic layers were evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 40:60) to give the title compound (1.7 g, 85.72%) as a yellow solid. LCMS: 184.2 (M+H-56).

手順：
５０ｍＬの一つ口丸底フラスコに、窒素下で（６－ホルミルスピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３２－２）（１．５ｇ、６．２７ｍｍｏｌ）及びＥｔＯＨ（１５ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホンヒドラジド（１．１６ｇ、６．２７ｍｍｏｌ）及び触媒量のＡｃＯＨ（０．２ｍＬ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注いだ。固体を濾過し、真空下で乾燥させ、白色の固体として表題化合物を得た（２．２ｇ、８６．１３％）。ＬＣＭＳ：４２５．５ （Ｍ＋１８）。 Synthesis of tert-butyl (6-((2-tosylhydrazono)methyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-32-3)

procedure:
In a 50 mL single neck round bottom flask under nitrogen was added tert-butyl (6-formylspiro[3.3]heptan-2-yl)carbamate (INX-SM-32-2) (1.5 g, 6. 27 mmol) and EtOH (15 mL). To this solution was added p-toluenesulfone hydrazide (1.16 g, 6.27 mmol) and a catalytic amount of AcOH (0.2 mL) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water. The solid was filtered and dried under vacuum to give the title compound as a white solid (2.2g, 86.13%). LCMS: 425.5 (M+18).

手順：
３５ｍＬのバイアルに、窒素下で（６－（（２－トシルヒドラゾノ）メチル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３２－３）（１．０ｇ、２．４５ｍｍｏｌ）及びジオキサン（１０ｍＬ）を充填した。この溶液に、（４－ホルミルフェニル）ボロン酸（０．３６ｇ、２．４５ｍｍｏｌ）及びＫ_２ＣＯ_３（０．５１ｇ、３．６８ｍｍｏｌ）を室温で添加し、１００℃で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、３０：７０）によって精製し、黄色の固体として表題化合物を得た（０．１６ｇ、１９．７９％）。ＬＣＭＳ：２７４．３（Ｍ＋Ｈ－５６）。 Synthesis of tert-butyl (6-(4-formylbenzyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-32-4)

procedure:
In a 35 mL vial, tert-butyl (6-((2-tosylhydrazono)methyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-32-3) (1.0 g, 2.45 mmol) and dioxane (10 mL). To this solution was added (4-formylphenyl)boronic acid (0.36 g, 2.45 mmol) and K ₂ CO ₃ (0.51 g, 3.68 mmol) at room temperature and stirred for an additional 2 hours at 100°C. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexanes, 30:70) to give the title compound as a yellow solid (0.16 g, 19.79%). LCMS: 274.3 (M+H-56).

手順：
３５ｍＬのバイアルに、（６－（４－ホルミルベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３２－４）（０．１６ｇ、０．４８ｍｍｏｌ）、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．１３ｇ、０．３４ｍｍｏｌ）、ＭｇＳＯ_４（０．２９ｇ、２．４３ｍｍｏｌ）及びＤＣＭ（４ｍＬ）を充填した。この溶液に、ＨＣｌＯ_４（０．４０ｇ、２．４３ｍｍｏｌ）を添加し、室温で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、真空上で濃縮した。粗製物を冷水で粉砕し、沈殿した固体を濾過し、真空下で乾燥させた。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)methyl)phenyl)-7-hydroxy-8b- (2-Hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4 ,5] Synthesis of indeno[1,2-d][1,3]dioxol-4-one (INX-SM-32)

procedure:
In a 35 mL vial, tert-butyl (6-(4-formylbenzyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-32-4) (0.16 g, 0.48 mmol), (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9, 10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0.13 g, 0.34 mmol), MgSO ₄ ( 0.29 g, 2.43 mmol) and DCM (4 mL). To this solution was added HClO ₄ (0.40 g, 2.43 mmol) and stirred for an additional 2 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated _NaHCO3 solution and concentrated on vacuum. The crude was triturated with cold water and the precipitated solid was filtered and dried under vacuum.

The crude product was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.1% FA in water, B = acetonitrile, A:B, 80:20), retention time 18. Purified by 54 min to give the R isomer as a white solid (0.045 g, 15.76%); LCMS: 588.4 (M+H) ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton assignment) : δ: 5.45 (s, 1H, acetal-H), 5.05 (d, J=4.8Hz, 1H, C16H).

７－ホルミル－５－オキサ－２－アザスピロ［３．４］オクタン－２－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３１－１）の合成

手順：
１００ｍＬの三つ口丸底フラスコに、窒素下で７－（ヒドロキシメチル）－５－オキサ－２－アザスピロ［３．４］オクタン－２－カルボン酸ｔｅｒｔ－ブチル（１．０ｇ、４．１１ｍｍｏｌ）及びＤＣＭ（２０ｍＬ）を充填した。この溶液に、デス・マーチンペルヨージナン（ＤＭＰ）（３．４０、８．２２ｍｍｏｌ）を室温で添加し、３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、ゴム状の固体として表題化合物を得た（０．８ｇ、７８．７１％）。^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：９．５９（ｓ， １Ｈ）， ４．０８－４．０４（ｍ， １Ｈ）， ３．８８－３．７０（ｍ， ５Ｈ）， ３．２１－３．１９（ｍ， １Ｈ）， ２．３７－２．２１（ｍ， ２Ｈ）， １．３６（ｓ， ９Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((5-oxa-2-azaspiro[3.4]octan-7-yl)methyl)phenyl)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2', Synthesis reaction scheme of 1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-31)

Synthesis of tert-butyl 7-formyl-5-oxa-2-azaspiro[3.4]octane-2-carboxylate (INX-SM-31-1)

procedure:
In a 100 mL three-neck round bottom flask under nitrogen, tert-butyl 7-(hydroxymethyl)-5-oxa-2-azaspiro[3.4]octane-2-carboxylate (1.0 g, 4.11 mmol) and DCM (20 mL). Dess-Martin periodinane (DMP) (3.40, 8.22 mmol) was added to this solution at room temperature and stirred for 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated NaHCO ₃ solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a gummy solid (0.8 g, 78.71%). ¹ H NMR (DMSO-d6) δ: 9.59 (s, 1H), 4.08-4.04 (m, 1H), 3.88-3.70 (m, 5H), 3.21-3 .19 (m, 1H), 2.37-2.21 (m, 2H), 1.36 (s, 9H).

手順：
５０ｍＬの一つ口丸底フラスコに、窒素下で７－ホルミル－５－オキサ－２－アザスピロ［３．４］オクタン－２－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３１－１）（０．８ｇ、４．０４ｍｍｏｌ）及びＥｔＯＨ（３０ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホニルヒドラジド（０．９２ｇ、４．９７ｍｍｏｌ）及び触媒量のＡｃＯＨを添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水中に注ぎ、固体を濾過し、真空下で乾燥させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、２０：８０）によって精製し、白色の固体として表題化合物を得た（０．７ｇ、５１．５６％）。ＬＣＭＳ：４１０．８（Ｍ＋Ｈ）^＋。 Synthesis of tert-butyl 7-((2-tosylhydrazono)methyl)-5-oxa-2-azaspiro[3.4]octane-2-carboxylate (INX-SM-31-2)

procedure:
In a 50 mL single neck round bottom flask under nitrogen, tert-butyl 7-formyl-5-oxa-2-azaspiro[3.4]octane-2-carboxylate (INX-SM-31-1) (0. 8 g, 4.04 mmol) and EtOH (30 mL). To this solution was added p-toluenesulfonyl hydrazide (0.92 g, 4.97 mmol) and a catalytic amount of AcOH and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the solid was filtered and dried under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 20:80) to give the title compound as a white solid (0.7 g, 51.56%). LCMS: 410.8 (M+H) ⁺ .

手順：
３５ｍＬのバイアルに、７－（（２－トシルヒドラゾノ）メチル）－５－オキサ－２－アザスピロ［３．４］オクタン－２－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３１－２）（０．７２ｇ、１．７６ｍｍｏｌ）及びジオキサン（１０ｍＬ）を窒素下で充填した。この溶液に、（４－ホルミルフェニル）ボロン酸（０．２６ｇ、１．７６ｍｍｏｌ）及びＫ_２ＣＯ_３（０．４８ｇ、３．５２ｍｍｏｌ）を室温で添加し、１１０℃で更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、１５：８５）によって精製し、白色の固体として表題化合物を得た（０．３０ｇ、５２．９６％）。ＬＣＭＳ：３３２．８（Ｍ＋Ｈ）^＋。 Synthesis of tert-butyl 7-(4-formylbenzyl)-5-oxa-2-azaspiro[3.4]octane-2-carboxylate (INX-SM-31-3)

procedure:
In a 35 mL vial, add tert-butyl 7-((2-tosylhydrazono)methyl)-5-oxa-2-azaspiro[3.4]octane-2-carboxylate (INX-SM-31-2) (0.72 g). , 1.76 mmol) and dioxane (10 mL) under nitrogen. To this solution was added (4-formylphenyl)boronic acid (0.26 g, 1.76 mmol) and K ₂ CO ₃ (0.48 g, 3.52 mmol) at room temperature and stirred for an additional hour at 110°C. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 15:85) to give the title compound as a white solid (0.30 g, 52.96%). LCMS: 332.8 (M+H) ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、７－（４－ホルミルベンジル）－５－オキサ－２－アザスピロ［３．４］オクタン－２－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３１－３）（０．３０ｇ、０．９０ｍｍｏｌ）、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシレドニゾロン）（０．３４ｇ、０．９０ｍｍｏｌ）、ＭｇＳＯ_４（０．５４ｇ、４．５２ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＨＣｌＯ_４（０．４５ｇ、４．５２ｍｍｏｌ）を添加し、室温で更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((5-oxa-2-azaspiro[3.4]octan-7-yl)methyl)phenyl)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2', Synthesis of 1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-31)

procedure:
In a 10 mL single-neck round bottom flask, add tert-butyl 7-(4-formylbenzyl)-5-oxa-2-azaspiro[3.4]octane-2-carboxylate (INX-SM-31-3) ( 0.30g, 0.90mmol), (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl -6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyrednisolone) (0 .34 g, 0.90 mmol), _MgSO4 (0.54 g, 4.52 mmol) and DCM (5 mL). To this solution was added HClO ₄ (0.45 g, 4.52 mmol) and stirred for an additional hour at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate.

The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.1% FA in water, B = ACN:MEOH:IPA (65:25:10) , retention time: 16.40 min to obtain R isomer as 0.022 g of white solid, 4.50%); LCMS: 591.3 (M+H) ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton Assignment): δ: 5.44 (s, 1H, acetal-H), 5.06 (d, J=4.8 Hz, 1H, C16H).

（３－ホルミロキセタン－３－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３３－１）の合成

手順：
１００ｍＬの三つ口丸底フラスコに、窒素下で（３－（ヒドロキシメチル）オキセタン－３－イル）カルバミン酸ｔｅｒｔ－ブチル（２．０ｇ、９．８４ｍｍｏｌ）及びＤＣＭ（２０ｍＬ）を充填した。この溶液に、デス・マーチンペルヨージナン（ＤＭＰ）（４．１７ｇ、９．８４ｍｍｏｌ）を０℃で添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をセライトを通して濾過し、ジエチルエーテルで洗浄した。組み合わせた有機層を真空下で蒸発させ、粗生成物を得た。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：４５：５５）によって精製し、黄色の固体として表題化合物を得た（２．０ｇ、定量的）。^１Ｈ ＮＭＲ （ＣＤＣｌ３） δ：９．８５（ｓ， １Ｈ）， ５．５０－５．４２ （ｍ，１Ｈ）， ５．１０－４．９４０（ｍ， １Ｈ）， ４．８６－４．８４（ｄ， ２Ｈ）， １．４７ （ｓ， ９Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminoxetan-3-yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5] Synthesis reaction scheme of indeno[1,2-d][1,3]dioxol-4-one (INX-SM-33)

Synthesis of tert-butyl (3-formyloxetan-3-yl)carbamate (INX-SM-33-1)

procedure:
A 100 mL three-neck round bottom flask was charged with tert-butyl (3-(hydroxymethyl)oxetan-3-yl)carbamate (2.0 g, 9.84 mmol) and DCM (20 mL) under nitrogen. Dess-Martin periodinane (DMP) (4.17 g, 9.84 mmol) was added to this solution at 0° C. and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was filtered through Celite and washed with diethyl ether. The combined organic layers were evaporated under vacuum to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane: 45:55) to give the title compound as a yellow solid (2.0 g, quantitative). ¹ H NMR (CDCl3) δ: 9.85 (s, 1H), 5.50-5.42 (m, 1H), 5.10-4.940 (m, 1H), 4.86-4.84 (d, 2H), 1.47 (s, 9H).

手順：
５０ｍＬの一つ口丸底フラスコに、窒素下で（３－ホルミロキセタン－３－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３３－１）（１．７ｇ、８．４４ｍｍｏｌ）及びＥｔＯＨ（１７ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホニルヒドラジド（１．５７ｇ、８．４４ｍｍｏｌ）及び触媒性ＡｃＯＨを添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、５０：５０）によって精製し、白色の固体として表題化合物を得た（２．５ｇ、８０．１０％）。ＬＣＭＳ：３８７．４（Ｍ＋１８）。 Synthesis of tert-butyl (3-((2-tosylhydrazono)methyl)oxetan-3-yl)carbamate (INX-SM-33-2)

procedure:
In a 50 mL single neck round bottom flask, tert-butyl (3-formyloxetan-3-yl)carbamate (INX-SM-33-1) (1.7 g, 8.44 mmol) and EtOH (17 mL) were added under nitrogen. filled with. To this solution was added p-toluenesulfonyl hydrazide (1.57 g, 8.44 mmol) and catalytic AcOH and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 50:50) to give the title compound as a white solid (2.5 g, 80.10%). LCMS: 387.4 (M+18).

手順：
５０ｍＬのバイアルに、窒素下で（３－（（２－トシルヒドラゾノ）メチル）オキセタン－３－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３３－２）（２．５ｇ、６．７７ｍｍｏｌ）及びジオキサン（２５ｍＬ）を充填した。この溶液に、（４－ホルミルフェニル）ボロン酸（１．０ｇ、６．７７ｍｍｏｌ）及びＫ_２ＣＯ_３（１．４ｇ、１０．１６ｍｍｏｌ）を室温で添加し、１００℃で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、３０：７０）によって精製し、黄色の固体として表題化合物を得た（０．２５ｇ、１２％）。ＬＣＭＳ：２９２．２（Ｍ＋Ｈ）^＋。 Synthesis of tert-butyl (3-(4-formylbenzyl)oxetan-3-yl)carbamate (INX-SM-33-3)

procedure:
In a 50 mL vial, tert-butyl (3-((2-tosylhydrazono)methyl)oxetan-3-yl)carbamate (INX-SM-33-2) (2.5 g, 6.77 mmol) and dioxane were added under nitrogen. (25 mL). To this solution was added (4-formylphenyl)boronic acid (1.0 g, 6.77 mmol) and K ₂ CO ₃ (1.4 g, 10.16 mmol) at room temperature and stirred for an additional 2 hours at 100°C. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 30:70) to give the title compound as a yellow solid (0.25 g, 12%). LCMS: 292.2 (M+H) ⁺ .

手順：
３５ｍＬのバイアルに、（３－（４－ホルミルベンジル）オキセタン－３－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３３－１）（０．０８０ｇ、０．２７ｍｍｏｌ）、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．０７３ｇ、０．１９ｍｍｏｌ）、ＭｇＳＯ_４（０．１６ｇ、１．３７ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＨＣｌＯ_４（０．２３ｇ、１．３７ｍｍｏｌ）を添加し、室温で更に４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、真空下で濃縮した。粗製物を冷水で粉砕し、沈殿した固体を濾過し、真空下で乾燥させた。粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．１％ＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ、８０：２０）、保持時間：８．７０分によって精製し、白色の固体として表題化合物のＲ異性体を得た（０．００４ｇ、２．６５％）ＬＣＭＳ：５５１．３（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：５．４８（ｓ， １Ｈ， アセタール－Ｈ）， ５．０７（ｄ， Ｊ＝５．４Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminoxetan-3-yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5] Indeno[1,2-d][1,3]dioxol-4-one (INX-SM-33)

procedure:
In a 35 mL vial, tert-butyl (3-(4-formylbenzyl)oxetan-3-yl)carbamate (INX-SM-33-1) (0.080 g, 0.27 mmol), (8S, 9S, 10R , 11S, 13S, 14S, 16R, 17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12, 13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0.073 g, 0.19 mmol), MgSO ₄ (0.16 g, 1. 37 mmol) and DCM (2 mL). To this solution was added HClO ₄ (0.23 g, 1.37 mmol) and stirred for an additional 4 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated _NaHCO3 solution and concentrated under vacuum. The crude was triturated with cold water and the precipitated solid was filtered and dried under vacuum. The crude product was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.1% FA in water, B = acetonitrile, A:B, 80:20), retention time: 8 Purified by .70 min to give the R isomer of the title compound as a white solid (0.004 g, 2.65%) LCMS: 551.3 (M+H) ⁺ ; ¹ H NMR (400 MHz, MeOD, major Proton assignment): δ: 5.48 (s, 1H, acetal-H), 5.07 (d, J=5.4Hz, 1H, C16H).

４－イソシアナトビシクロ［２．２．２］オクタン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－１０－１）の合成

手順：
５０ｍＬの一つ口丸底フラスコに、４－（メトキシカルボニル）ビシクロ［２．２．２］オクタン－１－カルボン酸（１ｇ、４．４７ｍｍｏｌ）及びトルエン（２０ｍＬ）を充填した。この溶液に、ジフェニルホスホリルアジド（ＤＰＰＡ）（１．２９ｇ、４．４７ｍｍｏｌ）及びトリエチルアミン（０．４７ｇ、４．４７ｍｍｏｌ）を添加した。反応混合物を１１０℃で２時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を室温で冷却し、酢酸エチルで希釈し、１０％クエン酸溶液、次いで、飽和重炭酸塩溶液で洗浄した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、１０：９０）によって精製し、無色の液体として表題化合物を得た。（０．４５ｇ、４５．４６％）。^１Ｈ ＮＭＲ （ＣＤＣｌ_３） δ：３．６２（ｓ， ３Ｈ）， １．９０－１．８７ （ｍ， １２Ｈ）。 6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((4-aminobicyclo[2.2.2]octan-1-yl)methyl)phenyl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis reaction scheme of :4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-10)

Synthesis of methyl 4-isocyanatobicyclo[2.2.2]octane-1-carboxylate (INX-SM-10-1)

procedure:
A 50 mL single neck round bottom flask was charged with 4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-carboxylic acid (1 g, 4.47 mmol) and toluene (20 mL). To this solution was added diphenylphosphoryl azide (DPPA) (1.29 g, 4.47 mmol) and triethylamine (0.47 g, 4.47 mmol). The reaction mixture was heated at 110°C for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was cooled to room temperature, diluted with ethyl acetate and washed with 10% citric acid solution and then saturated bicarbonate solution. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 10:90) to give the title compound as a colorless liquid. (0.45g, 45.46%). ^1H NMR ( _CDCl3 ) δ: 3.62 (s, 3H), 1.90-1.87 (m, 12H).

手順：
２５ｍＬの一つ口丸底フラスコに、４－イソシアナトビシクロ［２．２．２］オクタン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－１０－１）（０．４５ｇ、２．１５ｍｍｏｌ）及び６Ｎ ＨＣｌ（１０ｍＬ）を充填した。反応混合物を室温で更に１２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、表題化合物を含有する粗生成物を得た。粗製物をｎ－ペンテン及びジエチルエーテルで粉砕して、白色の固体を得た（０．４５ｇ、定量的）。^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：１２．２１（ｂｓ， １Ｈ）， ８．２０（ｓ， ３Ｈ）， １．８３－１．６９（ｍ， １２Ｈ）。 Synthesis of 4-aminobicyclo[2.2.2]octane-1-carboxylic acid (INX-SM-10-2)

procedure:
In a 25 mL single neck round bottom flask, add methyl 4-isocyanatobicyclo[2.2.2]octane-1-carboxylate (INX-SM-10-1) (0.45 g, 2.15 mmol) and 6N HCl. (10 mL). The reaction mixture was stirred for a further 12 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to obtain the crude product containing the title compound. The crude material was triturated with n-pentene and diethyl ether to give a white solid (0.45 g, quantitative). ^1H NMR (DMSO-d6) δ: 12.21 (bs, 1H), 8.20 (s, 3H), 1.83-1.69 (m, 12H).

手順：
２５ｍＬの三つ口丸底フラスコを窒素下でエタノール（５ｍＬ）で充填した。本溶液に、塩化チオニル（０．６２ｇ、５．３２ｍｍｏｌ）を０℃で添加し、４－アミノビシクロ［２．２．２］オクタン－１－カルボン酸（ＩＮＸ－ＳＭ－１０－２）（０．４５ｇ、２．６５ｍｍｏｌ）を添加し、３時間還流させた。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させて、粗生成物を得た。粗製物をｎ－ペンテン及びジエチルエーテルによる粉砕によって精製し、白色の固体として表題化合物を得た（０．５５ｇ、定量的）。ＬＣＭＳ：１９８．２０；^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：８．１３（ｓ， １Ｈ）， ７．６８（ｓ， ２Ｈ）， ４．０４－４．９９（ｑ， Ｊ＝６．８ Ｈｚ， ２Ｈ）， １．８２－１．７１（ｍ， １２ Ｈ） １．１６－１．１２（ｔ， ３Ｈ， Ｊ＝８ Ｈｚ）。 Synthesis of ethyl 4-aminobicyclo[2.2.2]octane-1-carboxylate (INX-SM-10-3)

procedure:
A 25 mL three neck round bottom flask was filled with ethanol (5 mL) under nitrogen. To this solution was added thionyl chloride (0.62 g, 5.32 mmol) at 0°C, and 4-aminobicyclo[2.2.2]octane-1-carboxylic acid (INX-SM-10-2) (0 .45 g, 2.65 mmol) was added and refluxed for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to obtain the crude product. The crude material was purified by trituration with n-pentene and diethyl ether to give the title compound as a white solid (0.55 g, quantitative). LCMS: 198.20; ^1H NMR (DMSO-d6) δ: 8.13 (s, 1H), 7.68 (s, 2H), 4.04-4.99 (q, J=6.8 Hz , 2H), 1.82-1.71 (m, 12 H) 1.16-1.12 (t, 3H, J=8 Hz).

手順：
２５ｍＬの三つ口丸底フラスコに、４－アミノビシクロ［２．２．２］オクタン－１－カルボン酸エチル（ＩＮＸ－ＳＭ－１０－３）（０．５５ｇ、２．２７ｍｍｏｌ）及びＴＨＦ（５．５ｍＬ）を窒素下で充填した。この溶液に、ＬｉＡｌＨ_４（ＴＨＦ中１Ｍ）（６．９ｍＬ、６．９ｍｍｏｌ）を－２０℃で添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を１０％ＮａＯＨ溶液でクエンチし、セライト床を通して濾過した。濾液をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をｎ－ペンテン及びジエチルエーテルで粉砕し、白色の固体として表題化合物を得た（０．３０ｇ、６９．３２％）。ＬＣＭＳ：１５６．１（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：３．０５（ｓ， ２Ｈ）， １．４８－１．３７（ｍ， １２Ｈ）。 Synthesis of (4-aminobicyclo[2.2.2]octan-1-yl)methanol (INX-SM-10-4)

procedure:
In a 25 mL three-neck round bottom flask, ethyl 4-aminobicyclo[2.2.2]octane-1-carboxylate (INX-SM-10-3) (0.55 g, 2.27 mmol) and THF (5 .5 mL) under nitrogen. To this solution was added LiAlH ₄ (1M in THF) (6.9 mL, 6.9 mmol) at −20° C. and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with 10% NaOH solution and filtered through a bed of Celite. The filtrate was dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was triturated with n-pentene and diethyl ether to give the title compound as a white solid (0.30 g, 69.32%). LCMS: 156.1 (M+H) ⁺ ; ¹ H NMR (DMSO-d6) δ: 3.05 (s, 2H), 1.48-1.37 (m, 12H).

手順：
２５ｍＬの一つ口丸底フラスコに、窒素下で（４－アミノビシクロ［２．２．２］オクタン－１－イル）メタノール（ＩＮＸ－ＳＭ－１０－４）（０．３０ｇ、１．９３ｍｍｏｌ）及びＤＣＭ（１５ｍＬ）を充填した。この溶液に、Ｂｏｃ－無水物（０．６３ｇ、２．９０ｍｍｏｌ）を室温で添加し、更に１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層を飽和重炭酸塩溶液で洗浄し、Ｎａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をジイソプロピルエーテルで粉砕し、白色の固体として表題化合物を得た（０．４５ｇ、９１．１９％）。ＬＣＭＳ：２００．２ （Ｍ＋Ｈ－５６）；^１Ｈ ＮＭＲ （ＣＤＣｌ３） δ：４．３４ （ｂｓ， １Ｈ）， ３．２７ （ｓ， ２Ｈ）， １．８６＝１．８２（ｍ， ６Ｈ）， １．５９－１．５３（ｍ， ６Ｈ）， １．４３（ｓ， ９Ｈ）。 Synthesis of tert-butyl (4-(hydroxymethyl)bicyclo[2.2.2]octan-1-yl)carbamate (INX-SM-10-5)

procedure:
In a 25 mL single neck round bottom flask under nitrogen, (4-aminobicyclo[2.2.2]octan-1-yl)methanol (INX-SM-10-4) (0.30 g, 1.93 mmol) and DCM (15 mL). To this solution was added Boc-anhydride (0.63 g, 2.90 mmol) at room temperature and stirred for an additional 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were washed with saturated bicarbonate solution, dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was triturated with diisopropyl ether to give the title compound as a white solid (0.45g, 91.19%). LCMS: 200.2 (M+H-56); ^1H NMR (CDCl3) δ: 4.34 (bs, 1H), 3.27 (s, 2H), 1.86=1.82 (m, 6H), 1.59-1.53 (m, 6H), 1.43 (s, 9H).

手順：
２５ｍＬの一つ口丸底フラスコに、（４－（ヒドロキシメチル）ビシクロ［２．２．２］オクタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１０－５）（０．４５ｇ、１．７６ｍｍｏｌ）及びＴＨＦ（１０ｍＬ）を充填した。デス・マーチンペルヨージナン（ＤＭＰ）（１．１２ｇ、２．６４ｍｍｏｌ）を室温で添加し、室温で１．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＮａＨＣＯ_３水溶液でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をブラインで洗浄し、Ｎａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、白色の固体として表題化合物を得た（０．４５ｇ、粗製）。ＬＣＭＳ：１９８．３（Ｍ＋Ｈ－５６）。 Synthesis of tert-butyl (4-formylbicyclo[2.2.2]octan-1-yl)carbamate (INX-SM-10-6)

procedure:
In a 25 mL single-neck round bottom flask, add tert-butyl (4-(hydroxymethyl)bicyclo[2.2.2]octan-1-yl)carbamate (INX-SM-10-5) (0.45 g, 1.76 mmol) and THF (10 mL). Dess Martin periodinane (DMP) (1.12 g, 2.64 mmol) was added at room temperature and stirred at room temperature for 1.5 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with aqueous NaHCO ₃ and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a white solid (0.45 g, crude). LCMS: 198.3 (M+H-56).

手順：
１０ｍＬのガラスバイアルに、（４－ホルミルビシクロ［２．２．２］オクタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１０－６）（０．４５ｇ、１．７７ｍｍｏｌ）及びエタノール（５ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホニルヒドラジド（０．３９ｇ、２．１３ｍｍｏｌ）及び酢酸（０．０５ｇ、０．８８ｍｍｏｌ）を室温で添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注いだ。白色の固体を濾過し、真空下で乾燥させ、オフホワイト色の固体として表題化合物を得た（０．３８ｇ、５１．４２％）。ＬＣＭＳ：４２２．３（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：１０．７２（ｓ， １Ｈ）， ７．６５（ｄ， Ｊ＝８．４ Ｈｚ， ２Ｈ）， ７．３９（ｄ， Ｊ＝８．０ Ｈｚ， ２Ｈ） ７．０２ （ｓ，１Ｈ）， ６．３７ （ｂｓ， １Ｈ）， ２．３７（ｓ， ３Ｈ）， １．７１－１．６９（ｍ， ６Ｈ）， １．４６－１．４２（ｍ， ６Ｈ），１．３４（ｓ， ９Ｈ）。 Synthesis of tert-butyl (4-((2-tosylhydrazono)methyl)bicyclo[2.2.2]octan-1-yl)carbamate (INX-SM-10-7)

procedure:
In a 10 mL glass vial, tert-butyl (4-formylbicyclo[2.2.2]octan-1-yl)carbamate (INX-SM-10-6) (0.45 g, 1.77 mmol) and ethanol ( 5 mL). To this solution, p-toluenesulfonyl hydrazide (0.39 g, 2.13 mmol) and acetic acid (0.05 g, 0.88 mmol) were added at room temperature and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water. The white solid was filtered and dried under vacuum to give the title compound as an off-white solid (0.38g, 51.42%). LCMS: 422.3 (M+H) ⁺ ; ¹ H NMR (DMSO-d6) δ: 10.72 (s, 1H), 7.65 (d, J=8.4 Hz, 2H), 7.39 (d , J=8.0 Hz, 2H) 7.02 (s, 1H), 6.37 (bs, 1H), 2.37 (s, 3H), 1.71-1.69 (m, 6H), 1.46-1.42 (m, 6H), 1.34 (s, 9H).

手順：
５０ｍＬの一つ口丸底フラスコに、（４－（（２－トシルヒドラゾノ）メチル）ビシクロ［２．２．２］オクタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１０－７）（１．０ｇ、２．３７ｍｍｏｌ）及びジオキサン（２０ｍＬ）を充填した。（４－ホルミルフェニル）ボロン酸（０．５３ｇ、３．５５ｍｍｏｌ）及びＫ_２ＣＯ_３（０．４９ｇ、３．５５ｍｍｏｌ）を室温で添加し、１１０℃で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：５０：５０）によって精製し、無色の液体として表題化合物を得た（０．０６ｇ、７．３６％）。ＬＣＭＳ：２８８．８（Ｍ＋Ｈ－５６）。 Synthesis of tert-butyl (4-(4-formylbenzyl)bicyclo[2.2.2]octan-1-yl)carbamate (INX-SM-10-8)

procedure:
In a 50 mL single neck round bottom flask, add tert-butyl (4-((2-tosylhydrazono)methyl)bicyclo[2.2.2]octan-1-yl)carbamate (INX-SM-10-7) ( 1.0 g, 2.37 mmol) and dioxane (20 mL). (4-formylphenyl)boronic acid (0.53 g, 3.55 mmol) and K ₂ CO ₃ (0.49 g, 3.55 mmol) were added at room temperature and stirred for an additional 2 hours at 110°C. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 50:50) to give the title compound as a colorless liquid (0.06 g, 7.36%). LCMS: 288.8 (M+H-56).

手順：
１０ｍＬの一つ口丸底フラスコに、（４－（４－ホルミルベンジル）ビシクロ［２．２．２］オクタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１０－８）（０．０５ｇ、０．１４５ｍｍｏｌ）及び（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシレドニゾロン）（０．０５４ｇ、０．１４ｍｍｏｌ）、ＭｇＳＯ_４（０．０８０ｇ、０．７３ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＨＣｌＯ_４（０．０７２ｇ、０．７３ｍｍｏｌ）を添加し、室温で更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和重炭酸塩溶液でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．１％ ＦＡ、Ｂ＝ＡＣＮ：ＭＥＯＨ：ＩＰＡ（６５：２５：１０）、Ａ：Ｂ、８０：２０）、保持時間１８．７６分によって精製し、白色の固体として表題化合物のＲ異性体を得た（０．０１５ｇ、１４．２７％）；ＬＣＭＳ ６０３．５２（Ｍ＋Ｈ）^＋、^１Ｈ ＮＭＲ（４００ＭＨｚ、ＭｅＯＤ主要プロトン割り当て） δ ：５．４６ （ｓ， １Ｈ， アセタール－Ｈ）， ５．０６ （ｄ， Ｊ＝４．８０ Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((4-aminobicyclo[2.2.2]octan-1-yl)methyl)phenyl)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1 Synthesis of ':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-10)

procedure:
In a 10 mL single neck round bottom flask, add tert-butyl (4-(4-formylbenzyl)bicyclo[2.2.2]octan-1-yl)carbamate (INX-SM-10-8) (0. 05g, 0.145mmol) and (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6 ,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyrednisolone) (0.054 g , 0.14 mmol), MgSO ₄ (0.080 g, 0.73 mmol) and DCM (5 mL). To this solution was added HClO ₄ (0.072 g, 0.73 mmol) and stirred for an additional hour at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated bicarbonate solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.1% FA in water, B = ACN:MEOH:IPA (65:25:10), A: B, 80:20), retention time 18.76 min to give the R isomer of the title compound as a white solid (0.015 g, 14.27%); LCMS 603.52 (M+H) ⁺ , ^1H NMR (400 MHz, MeOD major proton assignment) δ: 5.46 (s, 1H, acetal-H), 5.06 (d, J=4.80 Hz, 1H, C16H).

（Ｅ）－（３，３－ジフルオロ－１－（（２－トシルヒドラゾノ）メチル）シクロブチル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３５－１）の合成

手順：
３０ｍＬのガラスバイアルに、窒素下で（３，３－ジフルオロ－１－ホルミルシクロブチル）カルバミン酸ｔｅｒｔ－ブチル（０．５０ｇ、２．１２ｍｍｏｌ）及びジオキサン（５ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホニルヒドラジド（０．４ｇ、２．１２ｍｍｏｌ）を添加し、９０℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、３０：７０）によって精製し、薄黄色の固体として表題化合物を得た（０．５５ｇ、６４．２３％）。ＬＣＭＳ：３４８．１（Ｍ＋Ｈ－５６）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((1-amino-3,3-difluorocyclobutyl)methyl)phenyl)-7-hydroxy-8b- (2-Hydroxyacetyl)-6a,8a-dimethyl 1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4, 5] Synthesis reaction scheme of indeno[1,2-d][1,3]dioxol-4-one (INX-SM-35)

(E) Synthesis of tert-butyl-(3,3-difluoro-1-((2-tosylhydrazono)methyl)cyclobutyl)carbamate (INX-SM-35-1)

procedure:
A 30 mL glass vial was charged with tert-butyl (3,3-difluoro-1-formylcyclobutyl)carbamate (0.50 g, 2.12 mmol) and dioxane (5 mL) under nitrogen. To this solution was added p-toluenesulfonyl hydrazide (0.4 g, 2.12 mmol) and stirred at 90° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 30:70) to give the title compound as a pale yellow solid (0.55 g, 64.23%). LCMS: 348.1 (M+H-56).

手順：
３０ｍＬのバイアルに、窒素下で（３，３－ジフルオロ－１－（（２－トシルヒドラゾノ）メチル）シクロブチル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３５－１）（０．５０ｇ、１．７２ｍｍｏｌ）及びジオキサン（５ｍＬ）を充填した。この溶液に、（４－ホルミルフェニル）ボロン酸（０．１８ｇ、１．７２ｍｍｏｌ）及びＫ_２ＣＯ_３（０．２５ｇ、１．８５ｍｍｏｌ）を室温で添加し、１１０℃で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、１０：９０）によって精製し、白色の固体として表題化合物を得た（０．１１ｇ、２４．８０％）。ＬＣＭＳ：３２６．１（Ｍ＋Ｈ）^＋。 Synthesis of tert-butyl (3,3-difluoro-1-(4-formylbenzyl)cyclobutyl)carbamate (INX-SM-35-2)

procedure:
In a 30 mL vial, tert-butyl (3,3-difluoro-1-((2-tosylhydrazono)methyl)cyclobutyl)carbamate (INX-SM-35-1) (0.50 g, 1.72 mmol) was added under nitrogen. and dioxane (5 mL). To this solution was added (4-formylphenyl)boronic acid (0.18 g, 1.72 mmol) and K ₂ CO ₃ (0.25 g, 1.85 mmol) at room temperature and stirred for an additional 2 hours at 110°C. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 10:90) to give the title compound as a white solid (0.11 g, 24.80%). LCMS: 326.1 (M+H) ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（３，３－ジフルオロ－１－（４－ホルミルベンジル）シクロブチル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３５－３）（０．１１ｇ、０．３３ｍｍｏｌ）、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．１ｇ、０．２７ｍｍｏｌ）、ＭｇＳＯ_４（０．２ｇ、１．６９ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、ＨＣｌＯ_４（０．１６ｇ、１．６９ｍｍｏｌ）を添加し、室温で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％アンモニア、Ｂ＝アセトニトリル、Ａ：Ｂ、５８：４２）、保持時間１８．３６分によって精製し、白色の固体として表題化合物のＲ異性体（Ｆｒ－１）を得た（０．０３０ｇ、１５．５８％）；ＬＣＭＳ：５８５．４（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：５．４８（ｓ， １Ｈ， アセタール－Ｈ）， ５．０７（ｄ， Ｊ＝５．２Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((1-amino-3,3-difluorocyclobutyl)methyl)phenyl)-7-hydroxy-8b- (2-Hydroxyacetyl)-6a,8a-dimethyl 1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4, 5] Synthesis of indeno[1,2-d][1,3]dioxol-4-one (INX-SM-35)

procedure:
In a 25 mL single neck round bottom flask, add tert-butyl (3,3-difluoro-1-(4-formylbenzyl)cyclobutyl)carbamate (INX-SM-35-3) (0.11 g, 0.33 mmol). , (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9 ,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0.1 g, 0.27 mmol), MgSO ₄ (0.2 g, 1.69 mmol) and DCM (3 mL) were charged. To this solution was added HClO ₄ (0.16 g, 1.69 mmol) and stirred for an additional 2 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% ammonia in water, B = acetonitrile, A:B, 58: 42), purified by retention time 18.36 min to give the R isomer (Fr-1) of the title compound as a white solid (0.030 g, 15.58%); LCMS: 585.4 (M+H) ⁺ ; ^1H NMR (400 MHz, MeOD, major proton assignment): δ: 5.48 (s, 1H, acetal-H), 5.07 (d, J=5.2Hz, 1H, C16H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３，３－ジフルオロ－１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロブチル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１－１）の合成

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．２０ｇ、０．４１ｍｍｏｌ）、ＨＡＴＵ（０．２４ｇ、０．６４ｍｍｏｌ）、ＤＭＦ（２ｍＬ）及びＤＩＰＥＡ（０．１１ｇ、０．８２ｍｍｏｌ）を室温で充填した。この溶液に、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３，３－ジフルオロ－１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロブチル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３５）（０．２５ｇ、０．４１ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水：５０：５０）によって精製し、淡黄色の固体として表題化合物を得た（０．２４ｇ、５２．０３％）。 Synthesis of INX-A1

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3,3-difluoro-1-(4-((6aR,6bS) ,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8 , 8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl) Synthesis of tert-butyl (cyclobutyl)-5-oxopentanoate (INX-A1-1)

procedure:
In a 10 mL single neck round bottom flask, add (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5-oxopentanoic acid. (INX-P-4) (0.20 g, 0.41 mmol), HATU (0.24 g, 0.64 mmol), DMF (2 mL) and DIPEA (0.11 g, 0.82 mmol) were charged at room temperature. Add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3,3-difluoro-1-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- tert-Butyl (benzyl)cyclobutyl)-5-oxopentanoate (INX-SM-35) (0.25 g, 0.41 mmol) was added and stirred for 1 h at room temperature. After completion of the reaction as indicated by TLC. , the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO and evaporated _under vacuum to give the crude product. The crude was subjected to reverse phase column chromatography. (acetonitrile/water: 50:50) to give the title compound as a pale yellow solid (0.24 g, 52.03%).

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３，３－ジフルオロ－１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロブチル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１－１）（０．２ｇ、０．１２ｍｍｏｌ）及びＴＨＦ（３ｍＬ）を充填した。この溶液に、ジエチルアミン（０．３ｇ、０．２４ｍｍｏｌ）を室温で添加し、３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテル及びペンタンで粉砕して、黄色の固体として表題化合物を得た（０．１３ｇ、６８．７４％）ＬＣＭＳ：８２７．６ （Ｍ＋１）。 (S)-4-(2-aminoacetamide)-5-((3,3-difluoro-1-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7 -Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H- tert-butyl naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl)amino)-5-oxopentanoate (INX-A1 -2) Synthesis

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3,3-difluoro- 1-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2 ,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1, 3] Tert-butyl dioxol-10-yl)benzyl)cyclobutyl)-5-oxopentanoate (INX-A1-1) (0.2 g, 0.12 mmol) and THF (3 mL) were charged.To this solution, Diethylamine (0.3 g, 0.24 mmol) was added at room temperature and stirred for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether and pentane to give a yellow The title compound was obtained as a solid (0.13 g, 68.74%) LCMS: 827.6 (M+1).

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（３，３－ジフルオロ－１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロブチル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１－２）（０．１ｇ、０．１２ｍｍｏｌ）及びＤＣＭ（４ｍＬ）を充填した。この溶液に、水（１ｍＬ）中のＮａ_２ＣＯ_３（０．０４８ｇ、０．２４ｍｍｏｌ）溶液及びブロモアセチルブロミド（０．００５ｇ、０．４８ｍｍｏｌ）を室温で滴加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水：５０：５０）によって精製し、淡黄色の固体として表題化合物を得た（０．１０ｇ、６７．１０％）。ＬＣＭＳ：９４６．８， ８４８．９（Ｍ ＆ Ｍ＋２）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3,3-difluoro-1-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS , 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b -dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl)amino)-5-oxopentanoic acid tert- Synthesis of butyl (INX-A1-3)

procedure:
In a 10 mL single neck round bottom flask, (S)-4-(2-aminoacetamido)-5-((3,3-difluoro-1-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a, 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl)amino)-5- Tert-butyl oxopentanoate (INX-A1-2) (0.1 g, 0.12 mmol) and DCM (4 mL) were charged. To this solution was added a solution of Na ₂ CO ₃ (0.048 g, 0.24 mmol) in water (1 mL) and bromoacetyl bromide (0.005 g, 0.48 mmol) dropwise at room temperature and stirred for 1 h. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water: 50:50) to give the title compound as a pale yellow solid (0.10 g, 67.10%). LCMS: 946.8, 848.9 (M & M+2).

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３，３－ジフルオロ－１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロブチル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１－３）（０．１０ｇ、０．０１ｍｍｏｌ）を充填した。この溶液に、ＴＦＡ（０．２４ｇ、２．１０ｍｍｏｌ）を室温で添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た。（０．０９０ｇ、９５．６７％）。ＬＣＭＳ：８９０．９０， ８９３．０（Ｍ＆Ｍ＋２）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3,3-difluoro-1-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS , 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b -dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl)amino)-5-oxopentanoic acid (INX -Synthesis of A1-1)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3,3-difluoro-1-(4-()) in DCM (2 mL). (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- tert-butyl)benzyl)cyclobutyl)amino)-5-oxopentanoate (INX-A1-3) (0.10 g, 0.01 mmol) was charged. To this solution was added TFA (0.24 g, 2.10 mmol) at room temperature and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to give the title compound as an off-white solid. (0.090g, 95.67%). LCMS: 890.90, 893.0 (M&M+2).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチルの合成
（ＩＮＸ－Ｖ－１）

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．２５ｇ、０．４１ｍｍｏｌ）及びＨＡＴＵ（０．２０ｇ、０．４１ｍｍｏｌ）、ＤＭＦ（２ｍＬ）及びＤＩＰＥＡ（０．１０ｇ、０．８２ｍｍｏｌ）を室温で充填した。この溶液に、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－３２）（０．２５ｇ、０．４１ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水：５０：５０）によって精製し、淡黄色の固体として表題化合物を得た。これを即時に次のステップに使用した。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxo Synthesis reaction scheme of pentanoic acid (INX-V)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a ,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro)[3.3 ] Synthesis of tert-butyl heptan-2-yl)amino)-5-oxopentanoate (INX-V-1)

procedure:
In a 10 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Filled with oxopentanoic acid (INX-P-4) (0.25 g, 0.41 mmol) and HATU (0.20 g, 0.41 mmol), DMF (2 mL) and DIPEA (0.10 g, 0.82 mmol) at room temperature. did. Add (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)methyl)phenyl)- 7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2 ',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-32) (0.25 g, 0.41 mmol) was added and Stir for hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water: 50:50) to give the title compound as a pale yellow solid. This was used immediately for the next step.

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｖ－１）（０．２ｇ、０．１９ｍｍｏｌ）及びＴＨＦ（２ｍＬ）を充填した。この溶液に、ジエチルアミン（０．１４ｇ、１．９ｍｍｏｌ）を室温で添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテルで粉砕して、黄色の固体を得た（０．１５ｇ、９０．１２％）。ＬＣＭＳ：８３１．９（Ｍ＋Ｈ）^＋。 (S)-4-(2-aminoacetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-tert-butyl-5-oxopentanoate Synthesis of (INX-V-2)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- tert-butyl)benzyl)spiro)[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-V-1) (0.2 g, 0.19 mmol) and THF (2 mL). Filled. Diethylamine (0.14 g, 1.9 mmol) was added to this solution at room temperature and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether to give a yellow solid (0.15 g, 90.12%). LCMS: 831.9 (M+H) ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＣＭ（３ｍＬ）中の（Ｓ）－４－（２－アミノアセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｖ－２）（０．１５ｇ、０．２８ｍｍｏｌ）を充填した。この溶液に、水（１ｍＬ）中のＮａ_２ＣＯ_３（０．１１ｇ、０．５７ｍｍｏｌ）溶液、続いて、ブロモアセチルブロミド（０．０３７ｇ、０．１８ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水：５０：５０）によって精製し、淡黄色の固体として表題化合物を得た（０．０７０ｇ、４０％）。ＬＣＭＳ：９５０．９， ９５２．９（Ｍ ＆ Ｍ＋２）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxo Synthesis of tert-butyl pentanoate (INX-V-3)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-aminoacetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a, 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane tert-butyl-2-yl)amino)-5-oxopentanoate (INX-V-2) (0.15 g, 0.28 mmol) was charged. To this solution was added a solution of Na ₂ CO ₃ (0.11 g, 0.57 mmol) in water (1 mL) followed by bromoacetyl bromide (0.037 g, 0.18 mmol) at room temperature and stirred for 1 h. . After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water: 50:50) to give the title compound as a pale yellow solid (0.070 g, 40%). LCMS: 950.9, 952.9 (M & M+2).

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｖ－３）（０．０７０ｇ、０．０７ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．０５５ｇ、０．７１ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させて、薄黄色の固体として粗生成物を得た。粗製物を分取ＨＰＬＣ（カラム：Ｘｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ １９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．１％ＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ、５８：４２）によって精製し、Ｒ異性体を得て、これを保持時間１６．９２分で溶出させて、オフホワイト色の固体として表題化合物を得た（０．００４ｇ、１１．８３％）。ＬＣＭＳ：８９５．１ ＆ ８９７．１（Ｍ＆ Ｍ＋２）；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：５．４４（ｓ， １Ｈ， アセタール－Ｈ）， ５．０５（ｄ， Ｊ＝４．８Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxo Synthesis of pentanoic acid (INX-V)

procedure:
In a 10 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane- tert-Butyl (2-yl)amino)-5-oxopentanoate (INX-V-3) (0.070 g, 0.07 mmol) and DCM (2 mL) were charged. To this solution was added TFA (0.055 g, 0.71 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to obtain the crude product as a pale yellow solid. The crude was purified by preparative HPLC (column: Xbridge Prep, C18, OBD 19 x 250 mm, 5 μm, mobile phase: A = 0.1% FA in water, B = acetonitrile, A:B, 58:42), The R isomer was obtained which eluted with a retention time of 16.92 minutes to give the title compound as an off-white solid (0.004 g, 11.83%). LCMS: 895.1 & 897.1 (M&M+2); ^1H NMR (400 MHz, MeOD, major proton assignment): δ: 5.44 (s, 1H, acetal-H), 5.05 (d, J =4.8Hz, 1H, C16H).

Ｎ２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシル）－Ｎ６－（ｔｅｒｔ－ブトキシカルボニル）－Ｌ－リジン酸ベンジル（ＩＮＸ－Ｗ－１）の合成

手順：
２５０ｍＬの一つ口丸底フラスコに、（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシン（８．８ｇ、２９．６２ｍｍｏｌ）、ＨＡＴＵ（１６．９ｇ、４４．６７ｍｍｏｌ）、ＤＭＦ（１００ｍＬ）、及びＤＩＰＥＡ（１６ｇ、８９．２８ｍｍｏｌ）を室温で充填した。この溶液に、Ｎ６－（ｔｅｒｔ－ブトキシカルボニル）－Ｌ－リジン酸ベンジル（１０ｇ、２９．７６ｍｍｏｌ）を添加し、室温で４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をカラムクロマトグラフィー（酢酸エチル：ヘキサン、３０：７０）によって精製し、薄黄色の固体として表題化合物を得た（１５ｇ、８１．９６％）。ＬＣＭＳ：６１６．６（Ｍ＋Ｈ）^＋。 (S)-6-amino-2-(2-(2-bromoacetamido)acetamide)-N-(3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicycle[1.1.1]pentan-1-yl)hexane Synthetic reaction scheme of amide (INX-W)

Synthesis of N2-((((9H-fluoren-9-yl)methoxy)carbonyl)glycyl)-N6-(tert-butoxycarbonyl)-benzyl L-lysinate (INX-W-1)

procedure:
In a 250 mL single-necked round bottom flask, ((((9H-fluoren-9-yl)methoxy)carbonyl)glycine (8.8 g, 29.62 mmol), HATU (16.9 g, 44.67 mmol), DMF ( 100 mL) and DIPEA (16 g, 89.28 mmol) at room temperature.To this solution was added benzyl N6-(tert-butoxycarbonyl)-L-lysinate (10 g, 29.76 mmol) and Stirred for an hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was Purification by column chromatography (ethyl acetate:hexane, 30:70) gave the title compound as a pale yellow solid (15 g, 81.96%). LCMS: 616.6 (M+H) ⁺ .

手順：
１００ｍＬの一つ口丸底フラスコに、Ｎ２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシル）－Ｎ６－（ｔｅｒｔ－ブトキシカルボニル）－Ｌ－リジン酸ベンジル（ＩＮＸ－Ｗ－１）（５ｇ、８．１２ｍｍｏｌ）及びＭｅＯＨ（５０ｍＬ）を充填した。この溶液に、１０％Ｐｄ／Ｃ（２．５ｇ）を室温で添加し、水素で２時間パージした。ＴＬＣによって示される反応の完了後、反応混合物をセライト床を通して濾過し、濾液を真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、５０：５０）によって精製し、黄色の固体として表題化合物を得た（０．５ｇ、２３．４３％）。ＬＣＭＳ：４２６．２（Ｍ＋１－Ｂｏｃ）。 Synthesis of N2-((((9H-fluoren-9-yl)methoxy)carbonyl)glycyl)-N6-(tert-butoxycarbonyl)-L-lysine (INX-W-2)

procedure:
In a 100 mL one-neck round bottom flask, add benzyl N2-((((9H-fluoren-9-yl)methoxy)carbonyl)glycyl)-N6-(tert-butoxycarbonyl)-L-lysinate (INX-W- 1) (5 g, 8.12 mmol) and MeOH (50 mL) were charged. To this solution was added 10% Pd/C (2.5 g) at room temperature and purged with hydrogen for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was filtered through a bed of Celite and the filtrate was evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 50:50) to give the title compound as a yellow solid (0.5g, 23.43%). LCMS: 426.2 (M+1-Boc).

手順：
５０ｍＬの一つ口丸底フラスコに、Ｎ２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシル）－Ｎ６－（ｔｅｒｔ－ブトキシカルボニル）－Ｌ－リジン（ＩＮＸ－Ｗ－２）（０．５ｇ、０．９５ｍｍｏｌ）、ＨＡＴＵ（０．５４ｇ、１．４２ｍｍｏｌ）、ＤＭＦ（２５ｍＬ）、及びＤＩＰＥＡ（０．５ｍＬ、２．８５ｍｍｏｌ）を室温で充填した。この溶液に、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－３）（０．５３ｇ、０．９５ｍｍｏｌ）を添加し、室温で４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、７０：３０）によって精製し、黄色の固体として表題化合物を得た（０．４５ｇ、４４．３２％）。ＬＣＭＳ：１０６７．７（Ｍ＋Ｈ）^＋。 (2-(((S)-6-((tert-butoxycarbonyl)amino)-1-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino Synthesis of (9H-fluoren-9-yl)methyl)-1-oxohexan-2-yl)amino)-2-oxoethyl)carbamate (INX-W-3)

procedure:
In a 50 mL single-neck round bottom flask, add N2-((((9H-fluoren-9-yl)methoxy)carbonyl)glycyl)-N6-(tert-butoxycarbonyl)-L-lysine (INX-W-2). (0.5 g, 0.95 mmol), HATU (0.54 g, 1.42 mmol), DMF (25 mL), and DIPEA (0.5 mL, 2.85 mmol) were charged at room temperature. Add (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl) to this solution. phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H- Add naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-3) (0.53 g, 0.95 mmol), Stirred at room temperature for 4 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 70:30) to give the title compound as a yellow solid (0.45g, 44.32%). LCMS: 1067.7 (M+H) ⁺ .

手順：
５０ｍＬの一つ口丸底フラスコに、（２－（（（Ｓ）－６－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）－１－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－１－オキソヘキサン－２－イル）アミノ）－２－オキソエチル）カルバミン酸（９Ｈ－フルオレン－９－イル）メチル（ＩＮＸ－Ｗ－３）（０．４５ｇ、０．４２ｍｍｏｌ）及びＴＨＦ（２０ｍＬ）を充填した。この溶液に、ジエチルアミン（０．３０ｇ、４．２１ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で濃縮した。粗製物をジエチルエーテルで粉砕することによって精製し、真空下で乾燥させて、黄色の固体として表題化合物を得た（０．３５ｇ、９８．２３％）。ＬＣＭＳ：８４５．６（Ｍ＋Ｈ）^＋。 ((S)-5-(2-aminoacetamide)-6-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b- (2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2', 1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicycle[1.1.1]pentan-1-yl)amino)-6-oxohexyl) Synthesis of tert-butyl carbamate (INX-W-4)

procedure:
In a 50 mL single-neck round bottom flask, add (2-(((S)-6-((tert-butoxycarbonyl)amino)-1-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1 .1] (9H-fluoren-9-yl)methyl pentan-1-yl)amino)-1-oxohexan-2-yl)amino)-2-oxoethyl)carbamate (INX-W-3) (0. 45 g, 0.42 mmol) and THF (20 mL). To this solution was added diethylamine (0.30 g, 4.21 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated under vacuum. The crude was purified by trituration with diethyl ether and dried under vacuum to give the title compound as a yellow solid (0.35g, 98.23%). LCMS: 845.6 (M+H) ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（（Ｓ）－５－（２－アミノアセトアミド）－６－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクル［１．１．１］ペンタン－１－イル）アミノ）－６－オキソヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｗ－４）（０．３５ｇ、０．４１ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、水（１ｍＬ）中のＮａ_２ＣＯ_３（０．０７０ｇ、０．８２ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．１ｇ、０．４９ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、５０：５０）によって精製し、オフホワイト色の固体として表題化合物を得た（０．３３０ｇ、８２．４８％）。ＬＣＭＳ：９６７．５（Ｍ＋Ｈ）^＋。 ((S)-5-(2-(2-bromoacetamide)acetamide)-6-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7 -Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H- naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)- Synthesis of tert-butyl 6-oxohexyl)carbamate (INX-W-5)

procedure:
In a 25 mL single neck round bottom flask, ((S)-5-(2-aminoacetamide)-6-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS) , 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b -dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicycle[1.1.1]pentane-1- tert-butyl)amino)-6-oxohexyl)carbamate (INX-W-4) (0.35 g, 0.41 mmol) and DCM (5 mL) were charged. To this solution was added Na ₂ CO ₃ (0.070 g, 0.82 mmol) in water (1 mL) followed by bromoacetyl bromide (0.1 g, 0.49 mmol) at room temperature and stirred for 1 h. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 50:50) to give the title compound as an off-white solid (0.330g, 82.48%). LCMS: 967.5 (M+H) ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（（Ｓ）－５－（２－（２－ブロモアセトアミド）アセトアミド）－６－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－６－オキソヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｗ－５）（０．１０ｇ、０１０３ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＴＦＡ（０．０５９ｇ、０．５２ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗ＩＮＸ－Ｗを分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．１％ＦＡ、Ｂ＝ＡＣＮ：ＭｅＯＨ：ＩＰＡ（６５：２５：１０）、Ａ：Ｂ、６２：３８）、保持時間１９．０６分によって精製し、白色の固体としてＲ異性体を得た（０．００８ｇ、８．９３％）；^１Ｈ ＮＭＲ（４００ＭＨｚ、ＭｅＯＤ、主要プロトン割り当て） ： δ ：５．４７（ｓ， １Ｈ， アセタール－Ｈ）， ５．０７（ｄ， Ｊ＝４．８Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (S)-6-amino-2-(2-(2-bromoacetamido)acetamide)-N-(3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicycle[1.1.1]pentan-1-yl)hexane Synthesis of amide (INX-W)

procedure:
In a 10 mL single-necked round bottom flask, add ((S)-5-(2-(2-bromoacetamido)acetamide)-6-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a, 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1 ] tert-Butylpentan-1-yl)amino)-6-oxohexyl)carbamate (INX-W-5) (0.10 g, 0103 mmol) and DCM (5 mL) were charged. To this solution was added TFA (0.059 g, 0.52 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. Crude INX-W was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.1% FA in water, B = ACN:MeOH:IPA (65:25:10), A:B, 62:38), retention time 19.06 min to give the R isomer as a white solid (0.008 g, 8.93%); ¹ H NMR (400 MHz, MeOD, major proton Assignment): δ: 5.47 (s, 1H, acetal-H), 5.07 (d, J=4.8Hz, 1H, C16H).

（ｔｅｒｔ－ブトキシカルボニル）－Ｌ－アラニル－Ｌ－アラニン酸メチル（ＩＮＸ－Ｒ－１）の合成

手順：
３０ｍＬのガラスバイアルに、（ｔｅｒｔ－ブトキシカルボニル）－Ｌ－アラニン（５．０ｇ、２６．４５ｍｍｏｌ）、ＤＩＰＥＡ（１．３６ｍＬ、７９．３６ｍｍｏｌ）及びＤＭＦ（５０ｍＬ）を窒素下で充填した。この溶液に、ＨＡＴＵ（１５．０７ｇ、３９．６７ｍｍｏｌ）を０℃で添加し、続いて、Ｌ－アラニン酸塩メチル（３．６９ｇ、２６．４５ｍｍｏｌ）を添加した。反応混合物を室温で３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を氷冷水でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をヘキサン及びＤＣＭで粉砕し、白色の固体として表題化合物を得た（５．５ｇ、５６．２０％）。ＬＣＭＳ ２７５．３（Ｍ＋Ｈ）^＋。 (S)-2-(2-bromoacetamide)-N-((S)-1-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H -naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino) -1-oxopropan-2-yl)propanamide (INX-R) synthesis reaction scheme

Synthesis of (tert-butoxycarbonyl)-L-alanyl-L-methyl alaninate (INX-R-1)

procedure:
A 30 mL glass vial was charged with (tert-butoxycarbonyl)-L-alanine (5.0 g, 26.45 mmol), DIPEA (1.36 mL, 79.36 mmol) and DMF (50 mL) under nitrogen. To this solution was added HATU (15.07 g, 39.67 mmol) at 0° C. followed by methyl L-alaninate (3.69 g, 26.45 mmol). The reaction mixture was stirred at room temperature for 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with ice-cold water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was triturated with hexane and DCM to give the title compound as a white solid (5.5g, 56.20%). LCMS 275.3 (M+H) ⁺ .

手順：
１００ｍＬのガラス密封バイアルに、（ｔｅｒｔ－ブトキシカルボニル）－Ｌ－アラニル－Ｌ－アラニネート（ＩＮＸ－Ｒ－１）（４．５ｇ、１６．４２ｍｍｏｌ）及びＴＨＦ－水（９：１）（５５ｍＬ）を充填した。この溶液に、ＬｉＯＨ．Ｈ_２Ｏ（２０．６９ｇ、４９．２６ｍｍｏｌ）を添加し、６０℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をヘキサン及びＤＣＭで粉砕し、白色の固体として表題化合物を得た（４．０ｇ、９３．７０％）。ＬＣＭＳ：２６１．２０（Ｍ＋Ｈ）^＋。 Synthesis of (tert-butoxycarbonyl)-L-alanyl-L-alanine (INX-R2)

procedure:
In a 100 mL glass sealed vial, (tert-butoxycarbonyl)-L-alanyl-L-alaninate (INX-R-1) (4.5 g, 16.42 mmol) and THF-water (9:1) (55 mL) were added. Filled. This solution was added with LiOH. H ₂ O (20.69 g, 49.26 mmol) was added and stirred at 60° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was triturated with hexane and DCM to give the title compound as a white solid (4.0 g, 93.70%). LCMS: 261.20 (M+H) ⁺ .

手順：
３０ｍＬのガラスバイアルに、ＤＭＦ（５ｍＬ）及びＤＩＰＥＡ（０．６５ｍＬ、３．７８ｍｍｏｌ）中の（ｔｅｒｔ－ブトキシカルボニル）－Ｌ－アラニル－Ｌ－アラニン（ＩＮＸ－Ｒ－２）（０．３３ｇ、１．２６ｍｍｏｌ）を窒素下で充填した。この溶液に、ＨＡＴＵ（０．９６ｇ、２．５２ｍｍｏｌ）を０℃で添加し、続いて、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－３）（０．７０ｇ、１．２６ｍｍｏｌ）を添加した。結果として得られた反応混合物を室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を氷冷水でクエンチした。固体を濾過し、真空下で乾燥させた。粗製物をシリカゲルカラムクロマトグラフィー（メタノール／ＤＣＭ：６：９４）によって精製し、白色の固体として表題化合物を得た（０．３５ｇ、３４．４２％）。ＬＣＭＳ：８０２．６（Ｍ＋Ｈ）^＋。 ((S)-1-(((S)-1-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-1-oxopropane-2 Synthesis of tert-butyl (-yl)amino)-1-oxopropan-2-yl)carbamate (INX-R-3)

procedure:
In a 30 mL glass vial, (tert-butoxycarbonyl)-L-alanyl-L-alanine (INX-R-2) (0.33 g, 1 .26 mmol) under nitrogen. To this solution was added HATU (0.96 g, 2.52 mmol) at 0 °C, followed by (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-( (3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b, 7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-3) (0.70 g, 1.26 mmol) was added. The resulting reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with ice-cold water. The solid was filtered and dried under vacuum. The crude was purified by silica gel column chromatography (methanol/DCM: 6:94) to give the title compound as a white solid (0.35 g, 34.42%). LCMS: 802.6 (M+H) ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（（Ｓ）－１－（（（Ｓ）－１－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－１－オキソプロパン－２－イル）アミノ）－１－オキソプロパン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｒ－３）（０．３５ｇ、０．４４ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、ジエチルエーテル（３ｍｌ）中の２ＭのＨＣｌを添加し、室温で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテル及びｎ－ペンタンで粉砕して、黄色の固体として表題化合物を得た（０．３ｇ、９７．９２％）。ＬＣＭＳ：７０２．５ （Ｍ＋Ｈ）^＋。 (S)-2-amino-N-((S)-1-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b -(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-1-oxopropane Synthesis of -2-yl)propanamide (INX-R-4)

procedure:
In a 10 mL single neck round bottom flask, add ((S)-1-(((S)-1-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl ) amino)-1-oxopropan-2-yl) tert-butyl amino)-1-oxopropan-2-yl) carbamate (INX-R-3) (0.35 g, 0.44 mmol) and DCM (3 mL) ) was filled. To this solution was added 2M HCl in diethyl ether (3ml) and stirred for a further 2 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether and n-pentane to give the title compound as a yellow solid (0.3 g, 97.92%). LCMS: 702.5 (M+H) ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、窒素下で（Ｓ）－２－アミノ－Ｎ－（（Ｓ）－１－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－１－オキソプロパン－２－イル）プロパンアミド（ＩＮＸ－Ｒ－４）（０．３０ｇ、０．４３ｍｍｏｌ）及びＤＣＭ：水（８：２）（３．６ｍＬ）を充填した。この溶液に、Ｎａ_２ＣＯ_３（０．９１ｇ、０．８５５ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．８７ｇ、０．４３ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製の表題化合物ＩＮＸ－Ｒを分取ＨＰＬＣ（カラム：Ｘｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ １９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＮＨ_３、Ｂ＝アセトニトリル、Ａ：Ｂ、６５：３５）、保持時間２４．１０分によって精製し、白色の固体としてＲ異性体を得た（０．０３０ｇ、８．５３％）；ＬＣＭＳ：８２２．５， ８２４．４（Ｍ＆Ｍ＋２）；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：５．４６（ｓ， １Ｈ， アセタール－Ｈ）， ５．０５（ｄ， Ｊ＝５．２ Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (S)-2-(2-bromoacetamide)-N-((S)-1-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H -naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino) Synthesis of -1-oxopropan-2-yl)propanamide (INX-R)

procedure:
In a 10 mL single neck round bottom flask under nitrogen, (S)-2-amino-N-((S)-1-((3-(4-((6aR,6bS,7S,8aS,8bS,10R , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1] Pentan-1-yl)amino)-1-oxopropan-2-yl)propanamide (INX-R-4) (0.30 g, 0.43 mmol) and DCM:water (8:2) (3.6 mL) filled with. To this solution was added Na ₂ CO ₃ (0.91 g, 0.855 mmol) followed by bromoacetyl bromide (0.87 g, 0.43 mmol) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude title compound INX-R was purified by preparative HPLC (column: Xbridge Prep, C18, OBD 19 x 250 mm, 5 μm, mobile phase: A = 0.05% NH in water, _B = acetonitrile, A:B, 65: 35), purified by retention time 24.10 min to give the R isomer as a white solid (0.030 g, 8.53%); LCMS: 822.5, 824.4 (M&M+2); ¹ H NMR (400 MHz, MeOD, major proton assignment): δ: 5.46 (s, 1H, acetal-H), 5.05 (d, J=5.2 Hz, 1H, C16H).

ｔｅｒｔ－ブチル（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシル－Ｌ－アスパラギン酸メチルＩＮＸ－Ｘ－１）の合成

手順：
１００ｍＬのスクリューキャップガラスバイアルに、（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシン（５．０ｇ、１６．８３ｍｍｏｌ）、ＤＩＰＥＡ（８．６８ｍＬ、５０．５０ｍｍｏｌ）及びＤＭＦ（５０ｍＬ）を窒素下で充填した。この溶液に、ＨＡＴＵ（７．６７ｇ、２０．１９ｍｍｏｌ）、続いて、Ｌ－アスパラギン酸ｔｅｒｔ－ブチル（３．７９ｇ、２０．１９ｍｍｏｌ）を０℃で添加した。反応混合物を室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を氷冷水でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をヘキサン及びＤＣＭで粉砕し、白色の固体として表題化合物を得た（７．５ｇ、９５．４１％）。ＬＣＭＳ ４１２．８３（Ｍ－５６）。 (S)-2-(2-(2-bromoacetamide)acetamide)-N1-(3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)succinamide (INX-X ) synthesis reaction scheme

Synthesis of tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-methyl aspartate INX-X-1)

procedure:
In a 100 mL screw cap glass vial, (((9H-fluoren-9-yl)methoxy)carbonyl)glycine (5.0 g, 16.83 mmol), DIPEA (8.68 mL, 50.50 mmol) and DMF (50 mL) were added. Filled under nitrogen. To this solution was added HATU (7.67 g, 20.19 mmol) followed by tert-butyl L-aspartate (3.79 g, 20.19 mmol) at 0°C. The reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with ice-cold water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was triturated with hexane and DCM to give the title compound as a white solid (7.5 g, 95.41%). LCMS 412.83 (M-56).

手順：
２５０ｍＬの一つ口丸底フラスコに、ｔｅｒｔ－ブチル（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシル－Ｌ－アスパラギン酸メチル（ＩＮＸ－Ｘ－１）（２．０ｇ、４．２８ｍｍｏｌ）及びＤＣＭ（５０ｍＬ）を充填した。この溶液に、ＴＦＡ（４０ｍｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテル及びＤＣＭで粉砕して、白色の固体として表題化合物を得た（１．５ｇ、８５．２２％）。ＬＣＭＳ：４１２．８（Ｍ＋Ｈ）^＋。 Synthesis of (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-asparagine (INX-X2)

procedure:
In a 250 mL single neck round bottom flask, add methyl tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-aspartate (INX-X-1) (2.0 g, 4.28 mmol). ) and DCM (50 mL). To this solution was added TFA (40 ml) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether and DCM to give the title compound as a white solid (1.5 g, 85.22%). LCMS: 412.8 (M+H) ⁺ .

手順：
３０ｍＬのガラスバイアルに、窒素下で（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシル－Ｌ－アスパラギン（ＩＮＸ－Ｘ－２）（０．４ｇ、０．９７３ｍｍｏｌ）、ＤＭＦ（５ｍＬ）、ＨＡＴＵ（０．９６ｇ、２．５２ｍｍｏｌ）、及び（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－３）（０．７０ｇ、１．２６ｍｍｏｌ）を充填した。この溶液に、ＤＩＰＥＡ（０．５０ｍＬ、２．９１ｍｍｏｌ）を添加し、室温で３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を氷冷水でクエンチした。固体を濾過し、真空下で乾燥させた。粗製物をジエチルエーテル及びｎ－ペンタンで粉砕し、白色の固体として表題化合物を得た（０．６ｇ、６４．７２％）。ＬＣＭＳ：９５４．２４（Ｍ＋Ｈ）^＋。 (2-(((S)-4-amino-1-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-1,4-dioxobutane- Synthesis of (9H-fluoren-9-yl)methyl (2-yl)amino)-2-oxoethyl)carbamate (INX-X-3)

procedure:
In a 30 mL glass vial, (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-asparagine (INX-X-2) (0.4 g, 0.973 mmol), DMF (5 mL) under nitrogen. , HATU (0.96 g, 2.52 mmol), and (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1 ]Pentan-1-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-3) (0.70g , 1.26 mmol). To this solution was added DIPEA (0.50 mL, 2.91 mmol) and stirred at room temperature for 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with ice-cold water. The solid was filtered and dried under vacuum. The crude was triturated with diethyl ether and n-pentane to give the title compound as a white solid (0.6 g, 64.72%). LCMS: 954.24 (M+H) ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（２－（（（Ｓ）－４－アミノ－１－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－１，４－ジオキソブタン－２－イル）アミノ）－２－オキソエチル）カルバミン酸（９Ｈ－フルオレン－９－イル）メチル（ＩＮＸ－Ｘ－３）（０．３０ｇ、０．３１４ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ＤＥＡ（０．４８ｍＬ、４．７２ｍｍｏｌ）を添加し、室温で更に４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ヘキサンで粉砕して、黄色の固体として表題化合物を得た（０．２０ｇ、９６．０６％）。ＬＣＭＳ：７３１．０（Ｍ＋Ｈ）^＋。 (S)-2-(2-aminoacetamide)-N1-(3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2 -hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicycle[1.1.1]pentan-1-yl)succinamide (INX-X-4)

procedure:
In a 25 mL single neck round bottom flask, add (2-(((S)-4-amino-1-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl ) amino)-1,4-dioxobutan-2-yl)amino)-2-oxoethyl)(9H-fluoren-9-yl)methyl)carbamate (INX-X-3) (0.30 g, 0.314 mmol) and Charged with THF (5 mL). To this solution was added DEA (0.48 mL, 4.72 mmol) and stirred for an additional 4 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with hexane to give the title compound as a yellow solid (0.20 g, 96.06%). LCMS: 731.0 (M+H) ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、窒素下で（Ｓ）－２－（２－アミノアセトアミド）－Ｎ１－（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクル［１．１．１］ペンタン－１－イル）スクシンアミド（ＩＮＸ－Ｘ－４）（０．２０ｇ、０．２７３ｍｍｏｌ）及びＴＨＦ－水（８：２）（３．６ｍＬ）を充填した。この反応混合物に、Ｎａ_２ＣＯ_３（０．５８ｇ、０．５５ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０６６ｇ、０．３３ｍｍｏｌ）を添加し、室温で４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製の表題化合物ＩＮＸ－Ｘを分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＮＨ_３、Ｂ＝アセトニトリル、Ａ：Ｂ、６２：３８）、保持時間１７．７９分によって精製し、白色の固体としてＲ異性体を得た（０．０３０ｇ、８．５３％）；ＬＣＭＳ：８５２．７（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：５．４６（ｓ， １Ｈ， アセタール－Ｈ）， ５．０６（ｄ， Ｊ＝５．２ Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (S)-2-(2-(2-bromoacetamide)acetamide)-N1-(3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)succinamide (INX-X ) synthesis

procedure:
In a 25 mL single neck round bottom flask under nitrogen, (S)-2-(2-aminoacetamido)-N1-(3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicycle[1.1.1]pentane-1 -yl)succinamide (INX-X-4) (0.20 g, 0.273 mmol) and THF-water (8:2) (3.6 mL). To the reaction mixture was added Na ₂ CO ₃ (0.58 g, 0.55 mmol) followed by bromoacetyl bromide (0.066 g, 0.33 mmol) and stirred at room temperature for 4 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude title compound INX-X was purified by preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% NH in water, _B = acetonitrile, A:B, 62:38), retention time 17.79 min to give the R isomer as a white solid (0.030 g, 8.53%); LCMS: 852.7 (M+H) ⁺ ; ^1H NMR (400 MHz, MeOD, major proton assignment): δ: 5.46 (s, 1H, acetal-H), 5.06 (d, J=5.2 Hz, 1H, C16H).

（６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－６ｂ－フルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－Ｙ－１）の合成

手順：
１００ｍＬの一つ口丸底フラスコに、（８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－９－フルオロ－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（トリアムシノロン）（１．０ｇ、２．５３ｍｍｏｌ）及び（３－（４－ホルミルベンジル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３－５）（０．７６ｇ、２．５３ｍｍｏｌ）及びＤＣＭ（１０ｍＬ）を充填した。この溶液に、ＭｇＳＯ_４（１．５１ｇ、１２．６５ｍｍｏｌ）を添加し、室温で５分間撹拌した。ＨＣｌＯ_４（１．２ｇ、１２．６５ｍｍｏｌ）を反応混合物に添加し、室温で更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水、６０：４０）によって精製し、薄黄色として表題化合物を得た（０．５ｇ、３４．１４％）。ＬＣＭＳ：５７９．４（Ｍ＋Ｈ）^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-6b- Fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl) Synthetic reaction scheme of amino)-5-oxopentanoic acid (INX-Y)

(6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)-6b -Fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H- Synthesis of naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-Y-1)

procedure:
In a 100 mL single-neck round bottom flask, (8S, 9R, 10S, 11S, 13S, 14S, 16R, 17S)-9-fluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)- 10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (triamcinolone) (1.0 g , 2.53 mmol) and tert-butyl (3-(4-formylbenzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-5) (0.76 g, 2.53 mmol). 53 mmol) and DCM (10 mL). To this solution was added MgSO ₄ (1.51 g, 12.65 mmol) and stirred at room temperature for 5 minutes. HClO ₄ (1.2 g, 12.65 mmol) was added to the reaction mixture and stirred for an additional hour at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water, 60:40) to give the title compound as a pale yellow color (0.5 g, 34.14%). LCMS: 579.4 (M+H) ⁺

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．４２ｇ、０．８７ｍｍｏｌ）、ＨＡＴＵ（０．４９ｇ、１．３０ｍｍｏｌ）、ＤＭＦ（４ｍＬ）及びＤＩＰＥＡ（０．２２ｇ、１．７４ｍｍｏｌ）を室温で充填した。この溶液に、（６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－６ｂ－フルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－Ｙ－１）（０．５０ｇ、０．９７ｍｍｏｌ）を室温で添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水、５０：５０）によって精製し、薄黄色の固体として表題化合物を得た（０．６５ｇ、７１．４２％）。 (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((6aS,6bR,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a ,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol10-yl)benzyl)bicyclo[1 .1.1] Synthesis of tert-butyl pentan-1-yl)amino)-5-oxopentanoate (INX-Y-2)

procedure:
In a 50 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Filled with oxopentanoic acid (INX-P-4) (0.42g, 0.87mmol), HATU (0.49g, 1.30mmol), DMF (4mL) and DIPEA (0.22g, 1.74mmol) at room temperature. did. Add (6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl) to this solution. phenyl)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b- Dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-Y-1) (0.50 g, 0.97 mmol) was added at room temperature and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water, 50:50) to give the title compound as a pale yellow solid (0.65 g, 71.42%).

手順：
５０ｍＬの一つ口丸底フラスコに、ＴＨＦ（４ｍＬ）中の（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－６ｂ－フルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ｄｉｍｅｔｈｙｌ－４－ｏｘｏ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｙ－２）（０．６５ｇ、０．６２ｍｍｏｌ）を充填した。この溶液に、ジエチルアミン（０．４０ｇ、６４．２４ｍｍｏｌ）を室温で添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテルで粉砕して、黄色の固体として表題化合物を得た（０．４２ｇ、８４．８２％）。ＬＣＭＳ：８２１．４（Ｍ＋Ｈ）^＋。 (S)-4-(2-aminoacetamide)-5-((3-(4-((6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-6b-fluoro-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)-5-oxopentane Synthesis of tert-butyl acid (INX-Y-3)

procedure:
In a 50 mL single neck round bottom flask, (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(( 3-(4-((6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4 -oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d ][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-tert-butyl-5-oxopentanoate (INX-Y-2) (0.65 g, 0.62 mmol). Diethylamine (0.40 g, 64.24 mmol) was added to this solution at room temperature and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether to give the title compound as a yellow solid (0.42 g, 84.82%). LCMS: 821.4 (M+H) ⁺ .

手順：
５０ｍＬの一つ口丸底フラスコに、ＤＣＭ（１０ｍＬ）中の（Ｓ）－４－（２－アミノアセトアミド）－５－（（３－（４－（（６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－６ｂ－フルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｙ－３）（０．４２ｇ、０．５１ｍｍｏｌ）を充填した。この溶液に、水（１ｍｌ）に溶解したＮａ_２ＣＯ_３（０．１１ｇ、１．０２ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．１０ｇ、０．５１ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、６０：４０）によって精製し、淡黄色の固体として表題化合物を得た（０．２０ｇ、４１．４５％）。ＬＣＭＳ：９４２．０（Ｍ＋Ｈ）^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-6b- Fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl) Synthesis of tert-butyl amino)-5-oxopentanoate (INX-Y-4)

procedure:
In a 50 mL single neck round bottom flask, add (S)-4-(2-aminoacetamide)-5-((3-(4-((6aS,6bR,7S,8aS,8bS, 10R, 11aR, 12aS, 12bS)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a, 8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1 .1.1] tert-Butyl pentan-1-yl)-5-oxopentanoate (INX-Y-3) (0.42 g, 0.51 mmol) was charged into this solution, dissolved in water (1 ml). Na ₂ CO ₃ (0.11 g, 1.02 mmol) followed by bromoacetyl bromide (0.10 g, 0.51 mmol) were added at room temperature and stirred for 1 h. After completion of the reaction as indicated by TLC. The reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO and evaporated _under vacuum. The crude was purified by reverse phase column chromatography (acetonitrile:water, 60:40 ) to give the title compound as a pale yellow solid (0.20 g, 41.45%). LCMS: 942.0 (M+H) ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－６ｂ－フルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｙ－４）（０．２０ｇ、０．２０ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．１１ｇ、１．０１ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗ＩＮＸ－Ｙを分取ＨＰＬＣ（カラム：Ｘｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、３０×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．１％ギ酸、Ｂ＝ＡＣＮ：ＭｅＯＨ、５０：５０、Ａ：Ｂ、４７：５３）、保持時間１８．８３分によって精製し、白色の固体としてＲ異性体（Ｆｒ－１）を得た（０．０４０ｇ、２２．３７％）。ＬＣＭＳ：８８５．８（Ｍ＋Ｈ）^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ－ｄ６， 主要プロトン割り当て）： δ：５．４８（ｓ， １Ｈ， アセタール－Ｈ）， ５．０６（ｄ， Ｊ＝４．８Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-6b- Fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl) Synthesis of amino)-5-oxopentanoic acid (INX-Y)

procedure:
In a 10 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((6aS, 6bR, 7S, 8aS, 8bS, 10R) , 11aR, 12aS, 12bS) -6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b , 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1. 1.1] tert-butyl pentan-1-yl)amino)-5-oxopentanoate (INX-Y-4) (0.20 g, 0.20 mmol) and DCM (2 mL) were charged. To this solution was added TFA (0.11 g, 1.01 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. Crude INX-Y was subjected to preparative HPLC (column: Xbridge Prep, C18, 30 x 250 mm, 5 μm, mobile phase: A = 0.1% formic acid in water, B = ACN:MeOH, 50:50, A:B, 47 :53), retention time 18.83 min to give R isomer (Fr-1) as a white solid (0.040 g, 22.37%). LCMS: 885.8 (M+H) ⁺ ; ¹ H NMR (400 MHz, DMSO-d6, major proton assignment): δ: 5.48 (s, 1H, acetal-H), 5.06 (d, J=4 .8Hz, 1H, C16H).

（６Ｓ，８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－６，９－ジフルオロ－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（ＩＮＸ－Ｓ－１）の合成

手順：
２５ｍＬの一つ口丸底フラスコに、（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ，１０，１０－テトラメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（フルオシノロンアセトニド）（１．０ｇ）及び５０％水性ＨＢＦ_４（２０ｍｌ）を添加し、次いで、室温で更に１６時間撹拌した。ＴＬＣによって示される反応の完了後、固体を濾過し、水で洗浄し、真空下で乾燥させて、ＩＮＸ－Ｓ－１（１．０ｇ、定量的）を生じさせた。ＬＣＭＳ：４１３．３（Ｍ＋Ｈ）^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)- 2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a , 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentane- Synthesis reaction scheme of 1-yl)amino)-5-oxopentanoic acid (INX-S)

(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl- Synthesis of 6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (INX-S-1)

procedure:
In a 25 mL one-neck round bottom flask, add (2S, 6aS, 6bR, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a. ,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5] Indeno[1,2-d][1,3]dioxol-4-one (fluocinolone acetonide) (1.0 g) and ₅₀ % aqueous HBF (20 ml) were added, then for a further 16 hours at room temperature. Stirred. After completion of the reaction as indicated by TLC, the solid was filtered, washed with water, and dried under vacuum to yield INX-S-1 (1.0 g, quantitative). LCMS: 413.3 (M+H) ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、６Ｓ，８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－６，９－ジフルオロ－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（ＩＮＸ－Ｓ－１）（１．０ｇ、２．４２ｍｍｏｌ）及び（３－（４－ホルミルベンジル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（０．８０ｇ、２．６６ｍｍｏｌ）及びＤＣＭ（１０ｍＬ）を充填した。この溶液に、ＭｇＳＯ_４（１．４２ｇ、１２．１４ｍｍｏｌ）を添加し、室温で更に５分間撹拌した。ＨＣｌＯ_４（１．２ｇ、１２．１４ｍｍｏｌ）を添加し、室温で更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水、６０：４０）によって精製し、薄黄色として表題化合物を得た（０．６１ｇ、４２．２８％）。ＬＣＭＳ：５９６．４（Ｍ＋Ｈ）^＋． ((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl )-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b -Synthesis of dodecahydrone-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-S-2)

procedure:
In a 25 mL single neck round bottom flask, add 6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxy acetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (INX-S -1) (1.0 g, 2.42 mmol) and tert-butyl (3-(4-formylbenzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (0.80 g, 2.66 mmol) and DCM (10 mL). To this solution was added MgSO ₄ (1.42 g, 12.14 mmol) and stirred for an additional 5 minutes at room temperature. HClO ₄ (1.2 g, 12.14 mmol) was added and stirred for an additional hour at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water, 60:40) to give the title compound as a pale yellow color (0.61 g, 42.28%). LCMS: 596.4 (M+H) ⁺ .

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．４５ｇ、０．９３ｍｍｏｌ）、ＨＡＴＵ（０．５３ｇ、１．４０ｍｍｏｌ）、ＤＭＦ（４ｍＬ）及びＤＩＰＥＡ（０．２３ｇ、１．８６ｍｍｏｌ）を室温で充填した。この溶液に、（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－Ｓ－２）（０．６１ｇ、１．０２ｍｍｏｌ）を室温で添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水、５０：５０）によって精製し、薄黄色の固体として表題化合物を得た（０．４０ｇ、５６．１９％）。ＬＣＭＳ：１０６１．５（Ｍ＋Ｈ）^＋。 (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((2S,6aS,6bR,7S,8aS , 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7 , 8,8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl) Synthesis of tert-butyl (benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-S-3)

procedure:
In a 50 mL single neck round bottom flask, add (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5-oxopentanoic acid. (INX-P-4) (0.45 g, 0.93 mmol), HATU (0.53 g, 1.40 mmol), DMF (4 mL) and DIPEA (0.23 g, 1.86 mmol) were charged at room temperature. Add ((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl) to this solution. ) methyl) phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-S-2) (0.61g _{. _} and evaporated under vacuum. The crude was purified by reverse phase column chromatography (acetonitrile/water, 50:50) to give the title compound as a pale yellow solid (0.40 g, 56.19 %). LCMS: 1061.5 (M+H) ⁺ .

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｓ３）（０．４１ｇ、０．３９ｍｍｏｌ）及びＴＨＦ（４ｍＬ）を充填した。この溶液に、ジエチルアミン（０．２８ｇ、３．９１ｍｍｏｌ）を室温で添加し、３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、黄色の固体として表題化合物を得た（０．２６ｇ、８２．２４％）。ＬＣＭＳ：８３８．５（Ｍ＋Ｈ）^＋。 (S)-4-(2-aminoacetamide)-5-((3-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino )-Synthesis of tert-butyl-5-oxopentanoate (INX-S-4)

procedure:
In a 50 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-( (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ tert-butyl 1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-S3) (0.41 g, 0.39 mmol ) and THF (4 mL). Diethylamine (0.28 g, 3.91 mmol) was added to this solution at room temperature and stirred for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to give the title compound as a yellow solid (0.26 g, 82.24%). LCMS: 838.5 (M+H) ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の（Ｓ）－４－（２－アミノアセトアミド）－５－（（３－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｓ４）（０．２２ｇ、０．２６ｍｍｏｌ）を充填した。この溶液に、水（１ｍＬ）中のＮａ_２ＣＯ_３（０．１０ｇ、０．５３ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０３０ｇ、０．２８ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水、６０：４０）によって精製し、薄黄色として表題化合物を得た（０．１ｇ、３９．７２％）。ＬＣＭＳ：９６０．４（Ｍ＋Ｈ）^＋。 (S)-4-(2-(2-bromoacetamide)acetamide))-5-((3-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12, 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentane Synthesis of tert-butyl-1-yl)amino)-5-oxopentanoate (INX-S-5)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-aminoacetamido)-5-((3-(4-((2S,6aS,6bR,7S,8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7, 8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl ) tert-butyl bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-S4) (0.22 g, 0.26 mmol) was charged. To this solution was added Na ₂ CO ₃ (0.10 g, 0.53 mmol) in water (1 mL) followed by bromoacetyl bromide (0.030 g, 0.28 mmol) at room temperature and stirred for 1 h. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water, 60:40) to give the title compound as a pale yellow color (0.1 g, 39.72%). LCMS: 960.4 (M+H) ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド））－５－（（３－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｓ－５）（０．１０ｇ、０．１０ｍｍｏｌ）を充填した。この溶液に、ＴＦＡ（０．０５９ｇ、０．５２ｍｍｏｌ）を室温で添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で直接蒸発させた。粗ＩＮＸ－Ｓを分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．１％ギ酸、Ｂ＝アセトニトリル、Ａ：Ｂ、６５：３５）、保持時間１７．７分によって精製し、白色の固体としてＲ異性体を得た（０．０１１ｇ、１１．６８％）。ＬＣＭＳ：９０２．３， ９０４．３（Ｍ ＆ Ｍ＋２）。^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ－ｄ６， 主要プロトン割り当て）： δ：５．４５（ｓ， １Ｈ， アセタール－Ｈ）， ４．９５（ｄ， Ｊ＝４．８Ｈｚ， １Ｈ， Ｃ１６Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a , 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentane- Synthesis of 1-yl)amino)-5-oxopentanoic acid (INX-S)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide))-5-((3-(4-((2S,6aS, 6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4, 6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole Filled with tert-butyl-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-S-5) (0.10 g, 0.10 mmol) did. To this solution was added TFA (0.059 g, 0.52 mmol) at room temperature and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was directly evaporated under vacuum. Crude INX-S was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.1% formic acid in water, B = acetonitrile, A:B, 65:35), retention time Purification by 17.7 min gave the R isomer as a white solid (0.011 g, 11.68%). LCMS: 902.3, 904.3 (M & M+2). ^1H NMR (400 MHz, DMSO-d6, major proton assignment): δ: 5.45 (s, 1H, acetal-H), 4.95 (d, J=4.8Hz, 1H, C16H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｔ－１）の合成

手順：
１０ｍＬのバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｐ－５）（０．２０ｇ、０．１９ｍｍｏｌ）及びＤＭＦ（１ｍＬ）を充填した。この溶液に、１Ｈ－テトラゾール（０．１３７ｇ、１．９５０ｍｍｏｌ）及び（ｔＢｕＯ）_２ＰＮＥｔ_２（１．３ｇ、４．６８ｍｍｏｌ）を室温で添加し、室温で７９時間撹拌した。ＴＬＣによって示される反応の完了後、過酸化水素（１．３ｇ、４．６８ｍｍｏｌ）を溶液中に添加した。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、８０：２０）によって精製し、薄黄色の固体として表題化合物を得た（０．０７０ｇ、２９．４７％）。これを即時に次のステップに使用した。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H -naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino) Synthesis of -5-oxopentanoic acid (INX-T)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS ,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- Synthesis of tert-butyl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-T-1)

procedure:
In a 10 mL vial, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((6aR,6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8, 8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo [1.1.1] tert-Butyl pentan-1-yl)amino)-5-oxopentanoate (INX-P-5) (0.20 g, 0.19 mmol) and DMF (1 mL) were charged. 1H-tetrazole (0.137 g, 1.950 mmol) and (tBuO) ₂ PNEt ₂ (1.3 g, 4.68 mmol) were added to this solution at room temperature and stirred at room temperature for 79 hours. After completion of the reaction as indicated by TLC, hydrogen peroxide (1.3 g, 4.68 mmol) was added into the solution. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 80:20) to give the title compound as a pale yellow solid (0.070g, 29.47%). This was used immediately for the next step.

手順：
１０ｍＬのガラスバイアルに、ＴＨＦ（１ｍＬ）中の（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｔ－１）（０．０７０ｇ、０．０５７ｍｍｏｌ）を充填した。この溶液に、ジエチルアミン（０．０４２ｇ、０．５７ｍｍｏｌ）を室温で添加し、室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を濃縮した。粗製物をジエチルエーテル及びヘキサンによる粉砕によって精製し、淡黄色の固体として表題化合物を得た（０．３０ｇ、５２．４４％）。これを即時に次のステップに使用した。 (S)-4-(2-aminoacetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-(( di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl ) Synthesis of tert-butyl amino)-5-oxopentanoate (INX-T-2)

procedure:
In a 10 mL glass vial, (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4 -((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl -4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2 -d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-tert-butyl-5-oxopentanoate (INX-T-1) (0 .070g, 0.057mmol). To this solution was added diethylamine (0.042 g, 0.57 mmol) at room temperature and stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated. The crude was purified by trituration with diethyl ether and hexane to give the title compound as a pale yellow solid (0.30 g, 52.44%). This was used immediately for the next step.

手順：
１０ｍＬのガラスバイアルに、ＤＣＭ（１ｍＬ）中の（Ｓ）－４－（２－アミノアセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｔ－２）（０．０３０ｇ、０．０３０ｍｍｏｌ）を充填した。この溶液に、水（０．１ｍＬ）中のＮａ_２ＣＯ_３（０．００６ｇ、０．０６０ｍｍｏｌ）溶液、続いて、ブロモアセチルブロミド（０．００６ｇ、０．０３０ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として粗生成物を得た（０．０４０ｇ、粗製）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b- (2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1] Synthesis of tert-butyl pentan-1-yl)amino)-5-oxopentanoate (INX-T-3)

procedure:
In a 10 mL glass vial, (S)-4-(2-aminoacetamido)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8, 8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo [1.1.1] Tert-butyl pentan-1-yl)amino)-5-oxopentanoate (INX-T-2) (0.030 g, 0.030 mmol) was charged. To this solution was added a solution of Na ₂ CO ₃ (0.006 g, 0.060 mmol) in water (0.1 mL) followed by bromoacetyl bromide (0.006 g, 0.030 mmol) for 1 h at room temperature. Stirred. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product as an off-white solid (0.040 g, crude).

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＣＭ（１ｍＬ）中の（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｔ－３）（０．００１ｇ、０．００８９ｍｍｏｌ）を充填した。この溶液に、ＴＦＡ（０．００５ｇ、０．０４３ｍｍｏｌ）及び触媒トリイソプロピルシランを室温で添加し、２０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、黄色の固体として表題化合物を得た。（０．００６ｇ、７１％）。ＬＣＭＳ：９４６．２， ９４８．２（Ｍ ＆ Ｍ＋２）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H -naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino) Synthesis of -5-oxopentanoic acid (INX-T)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((6aR,6bS,7S)) in DCM (1 mL). , 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4 , 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3] tert-butyl dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-T-3) (0.001 g, 0.0089 mmol) Filled. To this solution was added TFA (0.005 g, 0.043 mmol) and catalyst triisopropylsilane at room temperature and stirred for 20 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to give the title compound as a yellow solid. (0.006g, 71%). LCMS: 946.2, 948.2 (M & M+2).

ＩＮＸ－Ａ－２の合成

手順：
ジクロロメタン／アセトニトリル（５００ｍＬ／１００ｍＬ）中の化合物ＩＮＸ－Ａ－１（３．０ｇ、７．６４ｍｍｏｌ、１．０当量）の溶液に、環状無水物（３．０ｇ、３０．５８ｍｍｏｌ、４．０当量）及びＤＭＡＰ（１．８ｇ、１５．２９ｍｍｏｌ、２．０当量）を添加した。反応混合物を室温で２時間撹拌させ、混合物を減圧下で濃縮した。残渣を、ＤＣＭ／ＭｅＯＨ（１０％～１５％）＋０．１％ＡｃＯＨで溶出されたシリカゲル上のカラムクロマトグラフィーによって精製し、化合物ＩＮＸ－Ａ－２（３．２ｇ、８５％）を白色の固体として得た。ＴＬＣ：ＤＣＭ／ＭｅＯＨ＝１０：１。Ｒ_ｆ（化合物１）＝０．４５。Ｒ_ｆ（化合物２）＝０．３０。ＬＣ－ＭＳ：（Ｍ＋Ｈ）^＋＝３９４．４０ Synthesis reaction scheme of INX-A

Synthesis of INX-A-2

procedure:
To a solution of compound INX-A-1 (3.0 g, 7.64 mmol, 1.0 eq.) in dichloromethane/acetonitrile (500 mL/100 mL) was added the cyclic anhydride (3.0 g, 30.58 mmol, 4.0 eq. ) and DMAP (1.8g, 15.29mmol, 2.0eq) were added. The reaction mixture was allowed to stir at room temperature for 2 hours and the mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (10%-15%) + 0.1% AcOH to give compound INX-A-2 (3.2 g, 85%) as a white solid. obtained as. TLC:DCM/MeOH=10:1. R _f (compound 1) = 0.45. R _f (compound 2) = 0.30. LC-MS: (M+H) ⁺ =394.40

手順：
ＮＭＰ（４ｍＬ）中のＩＮＸ－Ａ－２（２２０ｍｇ、０．４５ｍｍｏｌ）及びＩＮＸ－Ａ－３（２３０ｍｇ、０．６７ｍｍｏｌ）の溶液に、ＨＡＴＵ（３４２ｍｇ、０．９０ｍｍｏｌ）及びＤＩＰＥＡ（２３２ｍｇ、１．８ｍｍｏｌ）を添加した。混合物を室温で５時間撹拌した。混合物を分取ＨＰＬＣ（ＡＣＮ／Ｈ_２Ｏ、０．１％ＨＣＯＯＨ）によって精製して、ＩＮＸ－Ａ（１２２ｍｇ、３９％）を得た。ＬＣＭＳ： ［Ｍ＋Ｈ］^＋ ＝ ７０３；^１Ｈ ＮＭＲ （ＣＤＣｌ_３， ３００ＭＨｚ） （δ， ｐｐｍ） ７．２０ （ｄ， Ｊ ＝ ９．０ Ｈｚ， １ Ｈ）， ６．７３ （ｓ， ２ Ｈ）， ６．５２ （ｂｒ， １ Ｈ）， ６．３３ （ｄ， Ｊ ＝ ９．０ Ｈｚ， １ Ｈ）， ６．１１ （ｓ， １ Ｈ）， ４．９１ （ｑ， Ｊ ＝ １７．３ Ｈｚ， ２ Ｈ）， ４．３５ （ｄ ＝ ９．３ Ｈｚ， １ Ｈ）， ３．７６ － ３．４２ （ｍ， １０ Ｈ）， ３．０３ （ｍ， １ Ｈ）， ２．７９ （ｍ， ２ Ｈ）， ２．６５ － ２．５６ （ｍ， ３ Ｈ）， ２．４２ － ２．０６ （ｍ， ７ Ｈ）， １．８４ － １．６３ （ｍ， ３ Ｈ）， １．２２ （ｍ， １Ｈ）， １．０２ （ｓ， ３ Ｈ）， ０．９０ （ｄ， Ｊ ＝ ７．２ Ｈｚ， ３ Ｈ）．^１９Ｆ ＮＭＲ （ＣＤＣｌ_３） （δ， ｐｐｍ） －１６６．０９ （ｑ）。 Synthesis of INX-A

procedure:
To a solution of INX-A-2 (220 mg, 0.45 mmol) and INX-A-3 (230 mg, 0.67 mmol) in NMP (4 mL) was added HATU (342 mg, 0.90 mmol) and DIPEA (232 mg, 1. 8 mmol) was added. The mixture was stirred at room temperature for 5 hours. The mixture was purified by preparative HPLC (ACN/H ₂ O, 0.1% HCOOH) to yield INX-A (122 mg, 39%). LCMS: [M+H] ⁺ = 703; ¹ H NMR (CDCl ₃ , 300 MHz) (δ, ppm) 7.20 (d, J = 9.0 Hz, 1 H), 6.73 (s, 2 H), 6.52 (br, 1 H), 6.33 (d, J = 9.0 Hz, 1 H), 6.11 (s, 1 H), 4.91 (q, J = 17.3 Hz, 2 H), 4.35 (d = 9.3 Hz, 1 H), 3.76 - 3.42 (m, 10 H), 3.03 (m, 1 H), 2.79 (m, 2 H), 2.65 - 2.56 (m, 3 H), 2.42 - 2.06 (m, 7 H), 1.84 - 1.63 (m, 3 H), 1.22 (m , 1H), 1.02 (s, 3H), 0.90 (d, J = 7.2 Hz, 3H). ^19F NMR ( _CDCl3 ) (δ, ppm) -166.09 (q).

Unless otherwise specified, all reactions described below were performed under a dry nitrogen atmosphere. All major chemicals were used as received. All other commercially available materials such as solvents, reagents, and catalysts were used without further purification. The reaction was monitored by thin layer chromatography (TLC) using pre-coated Merck silica gel 60F254 aluminum sheets (Merck, Germany). Visualization of the TLC plates was achieved using ultraviolet light, ninhydrin spray, and iodine vapor. 1H NMR (400 MHz) was recorded on a Bruker Advancer-III HD FT-NMR spectrophotometer (Bruker, USA) and interpreted manually. Column chromatographic separations were carried out using 230-400 mesh, 100-200 mesh, and 60-120 mesh silica gel or C18 silica as the stationary phase using the appropriate mobile phase.
Below is the LCMS method used for analysis of the final target.
LCMS method 1
Column details: X-BRIDGE BEH 2.1 x 50mm 2.5μm
Machine details: Water Acquity UPLC-H class equipped with PDA and Acquity SQ detector, column temperature: 35 °C, autosampler temperature: 5 °C, mobile phase A: 0.1% formic acid in Milli-Q water (pH = 2.70), Mobile phase B: 0.1% formic acid in Milli-Q water: acetonitrile (10:90).
Mobile phase gradient details: T = 0 min (97% A, 3% B) flow rate: 0.8 mL/min, T = 0.75 min (97% A, 3% B) flow rate: 0.8 mL/min, T = Gradient (2% A, 98% B) up to 2.7 minutes Flow rate: 0.8 mL/min, T = Gradient (0% A, 100% B) up to 3 minutes Flow rate: 1 mL/min, T = 3 .5 min (0% A, 100% B) Flow rate: 1 mL/min, T = 3. Gradient (97% A, 3% B) flow rate to 51 min: 0.8 mL/min, T = 4 min (97% A, 3% B), flow rate: 0.8 mL/min, flow rate: 0.8 mL/min, electrophoresis time: 4 minutes, UV detection method: PDA.
Mass parameters:
Probe: ESI, mode of ionization: positive and negative, cone voltage: 30V and 10V, capillary voltage: 3.0KV, extractor voltage: 1V, Rf lens: 0.1V, source temperature: 120°C, desolvation Japanese temperature: 400°C. Cone gas flow rate: 100 L/hour, desolvation gas flow rate: 800 L/hour.
LCMS method 2:
Column details: X-BRIDGE BEH 2.1 x 50mm 2.5μm
Machine details: Waters Acquity Ultra performance LC equipped with PDA and fitted with QDA detector, column temperature: 35 °C, autosampler temperature: 5 °C, mobile phase A: 0.1% formic acid in Milli-Q water ( pH=2.70), mobile phase B: 0.1% formic acid in Milli-Q water: acetonitrile (10:90). Mobile phase gradient details: T = 0 min (97% A, 3% B) flow rate: 0.8 mL/min, T = 0.75 min (97% A, 3% B) flow rate: 0.8 mL/min, T = Gradient (2% A, 98% B) up to 2.7 minutes Flow rate: 0.8 mL/min, T = Gradient (0% A, 100% B) up to 3 minutes Flow rate: 1 mL/min, T = 3 .5 min (10% A, 100% B) Flow rate: 1 mL/min, T = 3. Gradient (97% A, 3% B) flow rate to 51 min: 0.8 mL/min, T = 4 min (97% A, 3% B), flow rate: 0.8 mL/min, running time: 4 min, UV detection method: PDA.
Mass parameters: probe: ESI, mode of ionization: positive and negative, cone voltage: 30V and 10V, capillary voltage: 0.8KV, extractor voltage: 1V, Rf lens: 0.1V, source temperature: 120 °C , probe temperature: 600°C, cone gas flow rate: default, desolvation gas flow rate: default.
LCMS method 3
Column details: Xtimate C18 4.6 x 50mm 5μm
Machine details: Waters Acquity Ultra performance LC connected to PDA and equipped with SQ detector, column temperature: 35°C, autosampler temperature: 5°C, mobile phase A: 5mM ammonium bicarbonate and (pH =7.35), mobile phase B: acetonitrile. Mobile phase gradient details: T = 0 min (97% A, 3% B) flow rate = 1.0 ml/min, T = 0.20 min (97% A, 3% B) flow rate = 1.0 ml/min, T = Gradient (20% A, 80% B) up to 2.70 min Flow rate = 1.0 ml/min, T = Gradient (0% A, 100% B) up to 3.0 min Flow rate = 1.2 ml/min , T = 3.50 min (0% A, 100% B) flow rate = 1.2 ml/min, T = 3.51 min (97% A, 3% B) flow rate = 1.0 ml/min, T = 4 End of electrophoresis at 0 minutes (97% A, 3% B) Flow rate = 1.0 ml/min, electrophoresis time: 4 minutes, UV detection method: PDA.
Mass parameters: probe: ESI, mode of ionization: positive and negative, cone voltage: 30 and 10V, capillary voltage: 3.0KV, extractor voltage: 2V, Rf lens: 0.1V, source temperature:: 120° C, probe temperature: 400°C, cone gas flow rate: 100 L/h, desolvation gas flow rate: 800 L/h.
LCMS method 4
Column details: Xtimate C18 4.6 x 150mm 5μm
Machine details: Waters 996 photodiode array detector equipped with Waters Micromass ZQ detector, column temperature: 35°C, autosampler temperature: 15°C, mobile phase A: 5mM ammonium acetate in Milli-Q water and 0.1 % formic acid (pH = 3.50), mobile phase B: methanol mobile phase gradient details: T = 0 min (90% A, 10% B), T = 7.0 min (10% A, 90% B), T = 9.0 min (0% A, 100% B), gradient to T = 14.00 min (0% A, 100% B), T = 14.01 min (90% A, 10% B) , end of electrophoresis at T=17 minutes (90% A, 10% B), flow rate: 1.0 mL/min, electrophoresis time: 17 minutes, UV detection method: PDA.
Mass parameters:
Probe: ESI, mode of ionization: positive and negative, cone voltage: 30 and 10V, capillary voltage: 3.0KV, extractor voltage: 2V, Rf lens: 0.1V, source temperature: 120 °C, probe Temperature: 400°C, cone gas flow rate: 100 L/hour, desolvation gas flow rate: 800 L/hour.
LCMS method 5
Column details: Sunfire C18 150 x 4.6mm, 3.5μm
Machine details: Agilent 1260 Infinity-II and G6125C (LC/MSD) mass detector, column temperature: 35°C, autosampler temperature: 15°C, mobile phase A: 5mM ammonium acetate and 0.1% in Milli-Q water. Formic acid (pH = 3.50), mobile phase B: methanol mobile phase gradient details: T = 0 min (90% A, 10% B), T = 7.0 min (10% A, 90% B), T = 9.0 min (0% A, 100% B), T = gradient to 14.00 min (0% A, 100% B), T = 14.01 min (90% A, 10% B), End of electrophoresis at T=17 minutes (90% A, 10% B), flow rate: 1.0 mL/min, electrophoresis time: 17 minutes, UV detection method: PDA.
Mass parameters:
Probe: MMI, mode of ionization: (ESI) positive and negative, fragment voltage: 30V and 70V, capillary voltage: 3000V, source gas temperature: 325°C, vaporizer temperature: 225°C, gas flow rate: 12L / Spray, atomizer: 50.
HPLC method 1:
Column details: Sunfire C18 (150mm x 4.6mm), 3.5μm
Machine details: Agilent Technologies. 1260 series, Infinity-II with PDA detector, column temperature: 35 °C, autosampler temperature: 15 °C, mobile phase A: 0.05% trifluoroacetic acid in Milli-Q water (pH = 2.2), mobile Phase B: Acetonitrile.
Mobile phase gradient details: T = 0 min (90% A, 10% B) flow rate: 1.0 mL/min, T = 7.0 min (10% A, 90% B) flow rate: 1.0 mL/min, T = Gradient (0% A, 100% B) up to 9.0 min Flow rate: 1.0 mL/min, T = Gradient (0% A, 100% B) up to 14 min Flow rate: 1.0 mL/min, T = 14.01 minutes (90% A, 10% B) flow rate: 1 mL/min, end of electrophoresis at T = 17 minutes (90% A, 10% B) flow rate: 1.0 mL/min, flow rate: 1.0 mL /min, electrophoresis time: 17 minutes, UV detection method: PDA.
HPLC method 2
Column details: Atlantis C18 (150 mm x 4.6 mm), 5.0 μm or Welch C18 (150 mm x 4.6 mm), 5.0 μm
Machine details: Waters Alliance e2695 with 2998 PDA detector, column temperature: 35 °C, autosampler temperature: 15 °C, mobile phase A: 0.1% ammonia in Milli-Q water (pH = 10.5), mobile phase B: Acetonitrile. Mobile phase gradient details: T = 0 min (90% A, 10% B) flow rate: 1.0 mL/min, T = 7.0 min (10% A, 90% B) flow rate: 1.0 mL/min, T = Gradient (0% A, 100% B) up to 9.0 min Flow rate: 1.0 mL/min, T = Gradient (0% A, 100% B) up to 14 min Flow rate: 1.0 mL/min, T = 14.01 minutes (90% A, 10% B) flow rate: 1 mL/min, end of electrophoresis at T = 17 minutes (90% A, 10% B) flow rate: 1.0 mL/min, flow rate: 1.0 mL /min, electrophoresis time: 17 minutes, UV detection method: PDA.
HPLC details: Waters Alliance e2695 with 2998 PDA detector, column details: Atlantis C18 (150 mm x 4.6 mm), 5.0 μm or Welch C18 (150 mm x 4.6 mm), 5.0 μm, mobile phase A: Milli-Q in water 0.1% ammonia (pH = 10.5), mobile phase B: acetonitrile, flow rate: 1.0 mL/min, running time: 17 min HPLC method 3
Column details: Agilent 1260 Infinity-II and G6125C (LC/MSD) mass detector, column temperature: 35°C, autosampler temperature: 15°C, mobile phase A: 5mM ammonium acetate in water + 0.1% formic acid (pH = 3.5), mobile phase B: MeOH. Mobile phase gradient details: T = 0 min (90% A, 10% B), T = 7.0 min (10% A, 90% B), T = 9 min (0% A, 100% B), T = gradient up to 14 min (0% A, 100% B), T = 14.1 min (90% A, 10% B), T = 17.0 min (90% A, 10% B). Flow rate: 1 mL/min, run time: 17 minutes. UV detection method: PDA. Mass parameters: probe: MMI, mode of ionization: (ESI) positive and negative, fragment voltage: 70 V and 30 V, capillary voltage: 3000 V, source gas temperature: 325 °C, vaporizer temperature: 225 °C, gas Flow rate: 12L/min, atomizer: 50.

（６－（４－ホルミルフェノキシ）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４３－１）の合成

手順：
１０ｍＬのガラスバイアルに、窒素下でＴＨＦ（０．７ｍＬ）中の（６－ヒドロキシスピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（０．０５０ｇ、０．２１ｍｍｏｌ）及び４－ヒドロキシベンズアルデヒド（０．０５３ｇ、０．４３ｍｍｏｌ）を充填した。この溶液に、トリフェニルホスフィン（０．０８６ｇ、０．３２ｍｍｏｌ）及びＤＩＡＤ（０．０６６ｇ、０．３２ｍｍｏｌ）を添加し、６５℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、８０：２０）によって精製し、白色の固体として表題化合物を得た（０．０２５ｇ、３５．９％）。ＬＣＭＳ：３３２．２ ［Ｍ＋Ｈ］^＋， ^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＯＳ－ｄ６） δ：９．８４ （ｓ， １Ｈ）， ７．８２ （ｄ， Ｊ＝８．８Ｈｚ， ２Ｈ）， ７．１０ （ｂｒ ｓ， １Ｈ）， ７．０１ （ｄ， Ｊ＝８．８ Ｈｚ， ２Ｈ）， ４．７２ （五重線， １Ｈ）， ３．９０－３．８０ （ｍ， １Ｈ）， ２．６０－２．４０ （ｍ， ４Ｈ）， ２．２５－１．９０ （ｍ， ４Ｈ）， １．３５ （ｓ， ９Ｈ）。 Synthesis of tert-butyl (6-(4-formylphenoxy)spiro[3.3]heptan-2-yl)carbamate (INX-SM-43-1)

Synthesis of tert-butyl (6-(4-formylphenoxy)spiro[3.3]heptan-2-yl)carbamate (INX-SM-43-1)

procedure:
In a 10 mL glass vial, tert-butyl (6-hydroxyspiro[3.3]heptan-2-yl)carbamate (0.050 g, 0.21 mmol) and 4-in THF (0.7 mL) under nitrogen were added. Hydroxybenzaldehyde (0.053g, 0.43mmol) was charged. Triphenylphosphine (0.086 g, 0.32 mmol) and DIAD (0.066 g, 0.32 mmol) were added to this solution and stirred at 65° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 80:20) to give the title compound as a white solid (0.025 g, 35.9%). LCMS: 332.2 [M+H] ⁺ , ¹ H NMR (400 MHz, DMOS-d6) δ: 9.84 (s, 1H), 7.82 (d, J=8.8Hz, 2H), 7.10 (br s, 1H), 7.01 (d, J=8.8 Hz, 2H), 4.72 (quintet, 1H), 3.90-3.80 (m, 1H), 2.60 -2.40 (m, 4H), 2.25-1.90 (m, 4H), 1.35 (s, 9H).

手順：
１０ｍＬのガラスバイアルに、ＤＣＭ（２ｍＬ）中の（６－（４－ホルミルフェノキシ）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４３－１）（０．１ｇ、０．３０ｍｍｏｌ）及び（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．１１３ｇ、０．３０ｍｍｏｌ）を充填した。この溶液に、ＭｇＳＯ_４（０．１８１ｇ、１．５０ｍｍｏｌ）、続いて、ＨＣｌＯ_４（０．４５９ｇ、４．５３ｍｍｏｌ）を添加し、室温で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８（２５０×２０）ｍｍ、５μｍ、移動相：Ａ＝水中の０．１％ＦＡ、Ｂ＝アセトニトリル＋１０％ＭＴＢＥ、Ａ：Ｂ＝８０：２０）、保持時間１７．１９分）によって精製し、白色の固体として表題化合物を得た（０．０１７ｇ、８．９１％）。ＬＣＭＳ：５９０．４ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４６（ｄ， Ｊ＝１０．０Ｈｚ，１Ｈ）， ７．３４（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．８１（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．２６（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４１ （ｓ， アセタール－Ｈ， １Ｈ）， ５．０４ （ｄ， Ｊ＝４．８ Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．７０－４．２５（ｍ， ４Ｈ）， ３．６７－３．６３（ｍ， １Ｈ）， ２．７０－１．６０（ｍ， １７Ｈ）， １．５１（ｓ， ３Ｈ）， １．２０－１．０３（ｍ， ２Ｈ）， ０．９９（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)oxy)phenyl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-43)

procedure:
In a 10 mL glass vial, add tert-butyl (6-(4-formylphenoxy)spiro[3.3]heptan-2-yl)carbamate (INX-SM-43-1) (0.00%) in DCM (2 mL). 1g, 0.30mmol) and (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6 ,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0.113 g, 0 .30 mmol). To this solution was added MgSO ₄ (0.181 g, 1.50 mmol) followed by HClO ₄ (0.459 g, 4.53 mmol) and stirred for an additional 2 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18 (250 x 20) mm, 5 μm, mobile phase: A = 0.1% FA in water, B = acetonitrile + 10% MTBE, A:B = 80: 20), retention time 17.19 min) to give the title compound as a white solid (0.017 g, 8.91%). LCMS: 590.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ 7.46 (d, J = 10.0 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H) , 6.81 (d, J=8.4Hz, 2H), 6.26 (d, J=10.0Hz, 1H), 6.04 (s, 1H), 5.41 (s, acetal-H, 1H), 5.04 (d, J=4.8 Hz, C16H, 1H), 4.70-4.25 (m, 4H), 3.67-3.63 (m, 1H), 2.70 -1.60 (m, 17H), 1.51 (s, 3H), 1.20-1.03 (m, 2H), 0.99 (s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ４－１）の合成

手順：
５０ｍＬの丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．８ｇ、１．６５ｍｍｏｌ）、ＤＩＰＥＡ（０．８５ｍＬ、４．９ｍｍｏｌ）及びＤＭＦ（８ｍＬ）を窒素下で充填した。この溶液に、ＨＡＴＵ（１．２６ｇ、３．３１ｍｍｏｌ）及び（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）オキシ）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－４３）（１．０７ｇ、１．８２ｍｍｏｌ）を０℃で添加した。反応混合物を室温で１時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物を氷冷水に注ぎ、沈殿した固体を濾過し、真空下で乾燥させた。粗固体をジエチルエーテル／ｎ－ペンタンで粉砕し、白色の固体として表題化合物を得た（１．３ｇ、７４．３８％）。ＬＣＭＳ：１０５４．４０ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxo Synthesis of pentanoic acid (INX-A4)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a ,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro)[3.3 ] Synthesis of tert-butyl heptane-2-yl)amino)-5-oxopentanoate (INX-A4-1)

procedure:
In a 50 mL round bottom flask, add (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5-oxopentanoic acid. (INX-P-4) (0.8 g, 1.65 mmol), DIPEA (0.85 mL, 4.9 mmol) and DMF (8 mL) were charged under nitrogen. To this solution were added HATU (1.26 g, 3.31 mmol) and (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3] heptan-2-yl)oxy)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12, 12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-43) (1.07g, 1.82 mmol) was added at 0°C. The reaction mixture was allowed to stir at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into ice-cold water and the precipitated solid was filtered and dried under vacuum. The crude solid was triturated with diethyl ether/n-pentane to give the title compound as a white solid (1.3 g, 74.38%). LCMS: 1054.40 [M+H] ⁺ .

手順：
１００ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ４－１）（１．３ｇ、１．２３ｍｍｏｌ）及びＴＨＦ（１５ｍＬ）を充填した。この溶液に、ＤＥＡ（２．５ｍｌ、２３．９７ｍｍｏｌ）を室温で添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテル及びＤＣＭで粉砕して、黄色の固体として表題化合物を得た（０．９ｇ、８８．２３％）。ＬＣＭＳ：８３２．３９［Ｍ＋Ｈ］^＋。 (S)-4-(2-aminoacetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-tert-butyl-5-oxopentanoate Synthesis of (INX-A4-2)

procedure:
In a 100 mL single-neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- tert-butyl)phenoxy)spiro)[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A4-1) (1.3 g, 1.23 mmol) and THF (15 mL). Filled. To this solution was added DEA (2.5 ml, 23.97 mmol) at room temperature and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether and DCM to give the title compound as a yellow solid (0.9 g, 88.23%). LCMS: 832.39 [M+H] ⁺ .

手順：
１００ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ４－２）（０．８ｇ、０．９６１ｍｍｏｌ）及びＤＣＭ－水（８：２、１２ｍＬ）を充填した。この溶液に、Ｎａ_２ＣＯ_３（０．３０ｇ、２．８８ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．２３ｇ、１．１５ｍｍｏｌ）を添加し、室温で４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルクロマトグラフィー（ＭｅＯＨ／ＤＣＭ、５：９５）によって精製し、黄色の固体の／として表題化合物を得た（０．４ｇ、４３．６５％）。ＬＣＭＳ：９５４．６１［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxo Synthesis of tert-butyl pentanoate (INX-A4-3)

procedure:
In a 100 mL single-neck round bottom flask, (S)-4-(2-aminoacetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino )-5-oxopentanoate (INX-A4-2) (0.8 g, 0.961 mmol) and DCM-water (8:2, 12 mL) were charged. To this solution was added Na ₂ CO ₃ (0.30 g, 2.88 mmol) followed by bromoacetyl bromide (0.23 g, 1.15 mmol) and stirred at room temperature for 4 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was purified by silica gel chromatography (MeOH/DCM, 5:95) to give the title compound (0.4 g, 43.65%) as a yellow solid. LCMS: 954.61 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ４－３）（０．４０ｇ、０．４２０ｍｍｏｌ）及びＤＣＭ（１０ｍＬ）を充填した。この溶液に、ＴＦＡ（４ｍＬ）を添加し、室温で更に４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ヘキサンで粉砕した。粗製物を分取ＨＰＬＣ（カラム：Ｘｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ １９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝７２：２８）、保持時間１６．３９分によって精製し、白色の固体として表題化合物を得た（０．００６ｇ、１．５９％）；ＬＣＭＳ：８９６．２ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４８（ｄ， Ｊ＝１０．０Ｈｚ，１Ｈ）， ７．３６（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．８３（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．２９（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ６．０６（ｓ， １Ｈ）， ５．４３（ｓ， アセタール－Ｈ， １Ｈ）， ５．００－３．５０（ｍ， １１Ｈ）， ２．９０－１．６０（ｍ， ２１Ｈ）， １．５３（ｓ， ３Ｈ）， １．２０－１．０３（ｍ， ２Ｈ）， ０．９１（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxo Synthesis of pentanoic acid (INX-A4)

procedure:
In a 25 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R) , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptane- tert-Butyl (2-yl)amino)-5-oxopentanoate (INX-A4-3) (0.40 g, 0.420 mmol) and DCM (10 mL) were charged. To this solution was added TFA (4 mL) and stirred for an additional 4 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with hexane. The crude product was subjected to preparative HPLC (column: Xbridge Prep, C18, OBD 19 x 250 mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 72:28), retention time 16 Purified by .39 min to give the title compound as a white solid (0.006 g, 1.59%); LCMS: 896.2 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ 7. 48 (d, J=10.0Hz, 1H), 7.36 (d, J=8.4Hz, 2H), 6.83 (d, J=8.4Hz, 2H), 6.29 (d, J =10.0Hz, 1H), 6.06 (s, 1H), 5.43 (s, acetal-H, 1H), 5.00-3.50 (m, 11H), 2.90-1.60 (m, 21H), 1.53 (s, 3H), 1.20-1.03 (m, 2H), 0.91 (s, 3H).

（６－（３－フルオロ－４－ホルミルフェノキシ）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４４－１）の合成

手順：
３５ｍＬのガラスバイアルに、窒素下で（６－ヒドロキシスピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（０．５０ｇ、２．１９ｍｍｏｌ）、２－フルオロ－４－ヒドロキシベンズアルデヒド（０．６１ｇ、４．３５ｍｍｏｌ）、トリフェニルホスフィン（０．８６ｇ、３．２９ｍｍｏｌ）及びＴＨＦ（７ｍＬ）を充填した。この溶液に、ＤＩＡＤ（０．６６ｇ、３．２９ｍｍｏｌ）を添加し、反応混合物を６５℃で２時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を順相カラムクロマトグラフィー（酢酸エチル／ヘキサン、２０：８０）によって精製し、白色の固体として表題化合物を得た（０．４０ｇ、５１．９７％）。ＬＣＭＳ：３５０．１［Ｍ＋Ｈ］^＋ (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)oxy)-2-fluorophenyl)- 7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2 Synthesis of ',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-44)

Synthesis of tert-butyl (6-(3-fluoro-4-formylphenoxy)spiro[3.3]heptan-2-yl)carbamate (INX-SM-44-1)

procedure:
In a 35 mL glass vial, tert-butyl (6-hydroxyspiro[3.3]heptan-2-yl)carbamate (0.50 g, 2.19 mmol), 2-fluoro-4-hydroxybenzaldehyde (0 .61 g, 4.35 mmol), triphenylphosphine (0.86 g, 3.29 mmol) and THF (7 mL). To this solution was added DIAD (0.66 g, 3.29 mmol) and the reaction mixture was heated at 65° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by normal phase column chromatography (ethyl acetate/hexane, 20:80) to give the title compound as a white solid (0.40 g, 51.97%). LCMS: 350.1 [M+H] ⁺

手順：
２５ｍＬの一つ口丸底フラスコに、ＤＣＭ（５ｍＬ）中の（６－（３－フルオロ－４－ホルミルフェノキシ）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４４－１）（０．２０ｇ、０．５７ｍｍｏｌ）及び（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（０．２１５ｇ、０．５７ｍｍｏｌ）を充填した。この溶液に、ＭｇＳＯ_４（０．３４ｇ、２．８６ｍｍｏｌ）及びＨＣｌＯ_４（０．５７ｇ、５．７２ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、真空下で濃縮した。次いで、粗製物を冷水で粉砕し、沈殿した固体を濾過し、真空下で乾燥させた。粗製物を逆相カラムクロマトグラフィー（水：アセトニトリル、６０：４０）によって精製し、白色の固体として表題化合物を得た（０．０２０ｇ、５．７５％）。ＬＣＭＳ：６０８．４ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：７．４６－７．４２（ｍ， ２Ｈ）， ６．６７－６．５５（ｍ， ２Ｈ）， ６．２５（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．２７（ｓ， １Ｈ）， ５．６４（ｓ， アセタール－Ｈ， １Ｈ）， ５．０４－５．０３ （ｄ， Ｊ＝４．８Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．６３－４．２９（ｍ， ４Ｈ）， ３．６４－３．６０（ｍ， １Ｈ）， ２．７０－１．７３（ｍ， １７Ｈ）， １．５０（ｓ， ３Ｈ）， １．３０－１．１２（ｍ， ２Ｈ）， ０．９２（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)oxy)-2-fluorophenyl)- 7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2 Synthesis of ',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-44)

procedure:
In a 25 mL single neck round bottom flask, add tert-butyl (6-(3-fluoro-4-formylphenoxy)spiro[3.3]heptan-2-yl)carbamate (INX-SM) in DCM (5 mL). -44-1) (0.20g, 0.57mmol) and (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl) -10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (0.215 g, 0 .57 mmol). To this solution were added MgSO ₄ (0.34 g, 2.86 mmol) and HClO ₄ (0.57 g, 5.72 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated _NaHCO3 solution and concentrated under vacuum. The crude was then triturated with cold water and the precipitated solid was filtered and dried under vacuum. The crude material was purified by reverse phase column chromatography (water:acetonitrile, 60:40) to give the title compound as a white solid (0.020g, 5.75%). LCMS: 608.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton assignment): δ: 7.46-7.42 (m, 2H), 6.67-6.55 (m, 2H ), 6.25 (d, J=10Hz, 1H), 6.27 (s, 1H), 5.64 (s, acetal-H, 1H), 5.04-5.03 (d, J=4 .8Hz, C16H, 1H), 4.63-4.29 (m, 4H), 3.64-3.60 (m, 1H), 2.70-1.73 (m, 17H), 1.50 (s, 3H), 1.30-1.12 (m, 2H), 0.92 (s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（３－フルオロ－４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ５－１）の合成

手順：
５０ｍＬの丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（１．０ｇ、２．０７ｍｍｏｌ）及びＤＭＦ（１０ｍＬ）を窒素下で充填し、０℃で冷却した。この溶液に、ＨＡＴＵ（１．５７ｇ、４．１４ｍｍｏｌ）、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）オキシ）－２－フルオロフェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－４４）（１．２５ｇ、２．０７ｍｍｏｌ）、次いで、ＤＩＰＥＡ（１．０ｍＬ、６．２２ｍｍｏｌ）を０℃で添加した。反応混合物を室温で１時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物を氷冷水に注いだ。結果として得られた固体を濾過し、真空下で乾燥させた。固体をジエチルエーテル及びｎ－ペンタンで粉砕し、白色の固体として表題化合物を得た（１．３ｇ、５９．０９％）。ＬＣＭＳ：１０７２．８［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(3-fluoro-4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS )-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino) Synthesis of -5-oxopentanoic acid (INX-A5)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(3-fluoro-4-((6aR,6bS,7S , 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a ,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[ 3.3] Synthesis of tert-butyl heptane-2-yl)amino)-5-oxopentanoate (INX-A5-1)

procedure:
In a 50 mL round bottom flask, add (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tertbutoxy)-5-oxopentanoic acid ( INX-P-4) (1.0 g, 2.07 mmol) and DMF (10 mL) were charged under nitrogen and cooled to 0°C. To this solution, HATU (1.57 g, 4.14 mmol), (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3] heptan-2-yl)oxy)-2-fluorophenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-44) ( 1.25 g, 2.07 mmol) then DIPEA (1.0 mL, 6.22 mmol) was added at 0<0>C. The reaction mixture was allowed to stir at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into ice-cold water. The resulting solid was filtered and dried under vacuum. The solid was triturated with diethyl ether and n-pentane to give the title compound as a white solid (1.3 g, 59.09%). LCMS: 1072.8 [M+H] ⁺ .

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（３－フルオロ－４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ５－１）（１．３ｇ、１．２１ｍｍｏｌ）及びＴＨＦ（１０ｍＬ）を充填した。この溶液に、ＤＥＡ（１．８ｍｌ、１７．２６ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテル及びＤＣＭで粉砕して、黄色の固体として表題化合物を得た（０．９ｇ、８７．３７％）。ＬＣＭＳ：８５０．５［Ｍ＋Ｈ］^＋。 (S)-4-(2-aminoacetamide)-5-((6-(3-fluoro-4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentane Synthesis of tert-butyl acid (INX-A5-2)

procedure:
In a 50 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(3-fluoro -4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4 , 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3] tert-butyl dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A5-1) (1.3 g, 1.21 mmol) and THF ( 10 mL). To this solution was added DEA (1.8 ml, 17.26 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether and DCM to give the title compound as a yellow solid (0.9 g, 87.37%). LCMS: 850.5 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（６－（３－フルオロ－４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ５－２）（０．９ｇ、１．０５ｍｍｏｌ）及びＤＣＭ－水（８：２、１２ｍＬ）を充填した。この溶液に、Ｎａ_２ＣＯ_３（０．３３ｇ、３．１７ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．２５ｇ、１．２７ｍｍｏｌ）を添加し、室温で４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルクロマトグラフィー（ＭｅＯＨ／ＤＣＭ、５：９５）によって精製し、黄色の固体として表題化合物を得た（０．５ｇ、４９．０１％）。ＬＣＭＳ：Ｃ_４８Ｈ_６２ ^７９ＢｒＦＮ_３Ｏ_１２に対する計算値（９７０．３５）、実測値９７０．４０［Ｍ＋Ｈ］^＋； (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(3-fluoro-4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS )-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino) Synthesis of tert-butyl-5-oxopentanoate (INX-A5-3)

procedure:
In a 25 mL single neck round bottom flask, (S)-4-(2-aminoacetamido)-5-((6-(3-fluoro-4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12, 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptane-2 tert-butyl-amino)-5-oxopentanoate (INX-A5-2) (0.9 g, 1.05 mmol) and DCM-water (8:2, 12 mL) were charged. To this solution was added Na ₂ CO ₃ (0.33 g, 3.17 mmol) followed by bromoacetyl bromide (0.25 g, 1.27 mmol) and stirred at room temperature for 4 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was purified by silica gel chromatography (MeOH/DCM, 5:95) to give the title compound as a yellow solid (0.5 g, 49.01%). LCMS: Calculated value _{for C48H6279BrFN3O12 (} 970.35), _actual ^value 970.40 [ _M +H] ⁺ ;

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（３－フルオロ－４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ５－３）（０．２ｇ、０．０．２０５ｍｍｏｌ）及びＤＣＭ（７ｍＬ）を充填した。この溶液に、ＴＦＡ（３ｍＬ）を添加し、室温で４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ヘキサンで粉砕した。粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝４５：５５、保持時間１２分）によって精製し、白色の固体として表題化合物を得た（０．００６ｇ、３．１８％）、
ＬＣＭＳ：Ｃ_４４Ｈ_５４ ^７９ＢｒＦＮ_３Ｏ_１２に対する計算値（９１４．２９）、実測値９１４．５［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４７－７．４２（ｍ， ２Ｈ）， ６．６８（ｄｄ， Ｊ＝８．４ ＆ ２Ｈｚ， １Ｈ）， ６．５９（ｄｄ， Ｊ＝１２．４ ＆ ２．４Ｈｚ， １Ｈ）， ６．２７（Ｊ＝１０．０ ＆ ２．０Ｈｚ，１Ｈ）， ６．０４（ｓ， １Ｈ）， ５．６５（ｓ， １Ｈ， アセタール－Ｈ）， ５．０３（ｄ， Ｊ＝５．２ Ｈｚ， １Ｈ， Ｃ１６Ｈ）， ４．６４－４．５５（ｍ，２Ｈ）， ４．４４－４．４３（ｍ， １Ｈ）， ４．３４－４．３０（ｍ， ２Ｈ）， ４．２２－４．１６（ｍ， １Ｈ）， ３．９５（ｄ， ２Ｈ）， ３．９２－３．９０（ｍ， ２Ｈ）， ２．８０－１．８０（ｍ， ２１Ｈ）， １．５１（ｓ， ３Ｈ）， １．２５－１．１５（ｍ， ２Ｈ）， １．００（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(3-fluoro-4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS )-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino) Synthesis of -5-oxopentanoic acid (INX-A5)

procedure:
In a 25 mL single neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(3-fluoro-4-((6aR,6bS,7S,8aS) ,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b , 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3. 3] tert-Butyl heptane-2-yl)amino)-5-oxopentanoate (INX-A5-3) (0.2 g, 0.0.205 mmol) and DCM (7 mL) were charged. To this solution was added TFA (3 mL) and stirred at room temperature for 4 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with hexane. The crude product was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 45:55, retention time 12 minutes) to give the title compound as a white solid (0.006g, 3.18%).
LCMS: Calculated value for C ₄₄ H ₅₄ ⁷⁹ BrFN ₃ O ₁₂ (914.29), actual value 914.5 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ 7.47-7.42 (m , 2H), 6.68 (dd, J=8.4 & 2Hz, 1H), 6.59 (dd, J=12.4 & 2.4Hz, 1H), 6.27 (J=10.0 & 2.0Hz, 1H), 6.04 (s, 1H), 5.65 (s, 1H, acetal-H), 5.03 (d, J=5.2 Hz, 1H, C16H), 4.64 -4.55 (m, 2H), 4.44-4.43 (m, 1H), 4.34-4.30 (m, 2H), 4.22-4.16 (m, 1H), 3 .95 (d, 2H), 3.92-3.90 (m, 2H), 2.80-1.80 (m, 21H), 1.51 (s, 3H), 1.25-1.15 (m, 2H), 1.00(s, 3H).

（３－（４－ホルミルベンジル）フェニル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｊ２－Ａ１）の合成

手順：
５０ｍｌの一つ口丸底フラスコに、４－（ブロモメチル）ベンズアルデヒド（０．５ｇ、２．５ｍｍｏｌ）及びＴＨＦ（５ｍｌ）を充填した。この溶液に、（３－（４，４，５，５－テトラメチル－１，３，２－ジオキサボロラン－２－イル）フェニル）カルバミン酸ｔｅｒｔ－ブチル（１．２ｇ、３．７ｍｍｏｌ）及び炭酸カリウム（０．８ｇ、６．２ｍｍｏｌ）を添加し、Ｎ_２で１５分間パージした。ＰｄＣｌ_２．ｄｐｐｆ－ＤＣＭ（０．３ｇ、０．１５ｍｍｏｌ）を反応混合物に添加し、８０℃で１６時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル：ヘキサン、５：９５）によって精製し、薄黄色の固体として表題化合物を得た（０．３２ｇ、４０．９５％）。ＬＣＭＳ：３１２．４［Ｍ＋Ｈ］^＋。 Synthesis of (INX-J2)

Synthesis of tert-butyl (3-(4-formylbenzyl)phenyl)carbamate (INX-J2-A1)

procedure:
A 50 ml single neck round bottom flask was charged with 4-(bromomethyl)benzaldehyde (0.5 g, 2.5 mmol) and THF (5 ml). To this solution were added tert-butyl (3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate (1.2 g, 3.7 mmol) and potassium carbonate. (0.8 g, 6.2 mmol) was added and purged with _N2 for 15 min. _PdCl2 . dppf-DCM (0.3 g, 0.15 mmol) was added to the reaction mixture and allowed to stir at 80° C. for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate:hexane, 5:95) to give the title compound as a pale yellow solid (0.32 g, 40.95%). LCMS: 312.4 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（３－（４－ホルミルベンジル）フェニル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｊ２－Ａ１）（０．３０ｇ、０．９６ｍｍｏｌ）を充填し、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロシキ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６α－ヒドロキシプレドニゾール）（０．３６ｇ、０．９６ｍｍｏｌ）をＤＣＭ（１０ｍＬ）に溶解させた。この溶液に、ＭｇＳＯ_４（０．５７ｇ、４．８ｍｍｏｌ）を添加し、室温で５分間撹拌した。ＨＣｌＯ_４（０．４８ｇ、４．８２ｍｍｏｌ）を反応混合物に添加し、室温で更に３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＮａＨＣＯ_３溶液でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＶＩＲＤＩＳ分取シリカ、２ＥＰ－ＯＢＤ、２５０×１９ｍｍ Ｓ－５μｍ、移動相：Ａ＝ヘプタン中の０．１％アンモニア、Ｂ＝ＩＰＡ：ＭＴＢＥ（７０：３０）、Ａ：Ｂ、８０：２０）によって精製し、異性体１及び異性体２を得た。これらの異性体を、保持時間２５．４９分（ＩＮＸ－Ｊ２－Ｓ）及び３１．５３分（ＩＮＸ－Ｊ２－Ｒ）で溶出した。
ＩＮＸ－Ｊ２－Ｒ（異性体２、Ｒ異性体）：（収率：０．１０ｇ、１８．２％）。ＬＣＭＳ：５７０．４ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４５（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ７．３５（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７，２０（ｄ， Ｊ＝７．６Ｈｚ， ２Ｈ）， ７．００（ｔ， １Ｈ）， ６．５６－６．５２（ｍ， ３Ｈ）， ６．２７（ｄ， Ｊ＝１０Ｈｚ， Ｈ）， ６．０３（ｓ， １Ｈ）， ５．４４（ｓ， １Ｈ， アセタール－Ｈ）， ５．０６（ｄ， Ｊ＝５．２Ｈｚ， １Ｈ， Ｃ１６Ｈ）， ４．６３（ｄ， Ｊ＝１９．２Ｈｚ， １Ｈ）， ４．４３（ｂｒｓ， １Ｈ）， ４．３３（ｄ， Ｊ＝１９．２Ｈｚ， １Ｈ），３．８５（ｓ， ２Ｈ）， ２．７０－１．６００（ｍ， ９Ｈ）， １．５１（ｓ， ３Ｈ）， １．２０－１．００（ｍ， ２Ｈ）， ０．９３（ｓ， ３Ｈ）。
ＩＮＸ－Ｊ２－Ｓ（異性体１、Ｓ異性体）：（収率：０．０１０ｇ、１．８２％）。ＬＣＭＳ ５７０．４［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４９（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ７．２３－７．１７（ｍ， ４Ｈ）， ７．０１（ｔ， １Ｈ）， ６．５７－６．５２（ｍ， ３Ｈ）， ６．２７（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．１２（ｓ， アセタール－Ｈ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４０（ｄ， Ｊ＝６．４Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．４３（ｂｒ ｓ， １Ｈ）， ４．３３（ｄ， Ｊ＝１９．２Ｈｚ， １Ｈ）， ４．１２（ｄ， Ｊ＝１９．２Ｈｚ， １Ｈ）， ３．８５（ｓ， ２Ｈ）， ２．７０－１．６０（ｍ， ９Ｈ）， １．５２（ｓ， ３Ｈ）， １．２０－１．００（ｍ， ２Ｈ）， ０．９３（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-(3-aminobenzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a -dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d ][1,3]dioxol-4-one (INX-J2-R), and (6aR, 6bS, 7S, 8aS, 8bS, 10S, 11aR, 12aS, 12bS)-10-(4-(3-aminobenzyl) ) phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H -Synthesis of naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-J2-S)

procedure:
A 10 mL single-necked round bottom flask was charged with tert-butyl (3-(4-formylbenzyl)phenyl)carbamate (INX-J2-A1) (0.30 g, 0.96 mmol), and (8S, 9S ,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12 ,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16α-hydroxyprednisole) (0.36 g, 0.96 mmol) was dissolved in DCM (10 mL). . To this solution was added MgSO ₄ (0.57 g, 4.8 mmol) and stirred at room temperature for 5 minutes. HClO ₄ (0.48 g, 4.82 mmol) was added to the reaction mixture and stirred for an additional 3 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with NaHCO ₃ solution and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was subjected to preparative HPLC (column: VIRDIS preparative silica, 2EP-OBD, 250 x 19 mm S-5 μm, mobile phase: A = 0.1% ammonia in heptane, B = IPA:MTBE (70:30), A:B, 80:20) to give isomer 1 and isomer 2. These isomers eluted with retention times of 25.49 minutes (INX-J2-S) and 31.53 minutes (INX-J2-R).
INX-J2-R (isomer 2, R isomer): (Yield: 0.10 g, 18.2%). LCMS: 570.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ: 7.45 (d, J = 10 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7,20 (d, J=7.6Hz, 2H), 7.00 (t, 1H), 6.56-6.52 (m, 3H), 6.27 (d, J=10Hz, H), 6.03 (s, 1H), 5.44 (s, 1H, acetal-H), 5.06 (d, J=5.2Hz, 1H, C16H), 4.63 (d, J=19.2Hz , 1H), 4.43 (brs, 1H), 4.33 (d, J=19.2Hz, 1H), 3.85 (s, 2H), 2.70-1.600 (m, 9H), 1.51 (s, 3H), 1.20-1.00 (m, 2H), 0.93 (s, 3H).
INX-J2-S (isomer 1, S isomer): (Yield: 0.010 g, 1.82%). LCMS 570.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ: 7.49 (d, J=10 Hz, 1H), 7.23-7.17 (m, 4H), 7.01 (t, 1H), 6.57-6.52 (m, 3H), 6.27 (d, J=10Hz, 1H), 6.12 (s, acetal-H, 1H), 6.04 (s , 1H), 5.40 (d, J=6.4Hz, C16H, 1H), 4.43 (br s, 1H), 4.33 (d, J=19.2Hz, 1H), 4.12( d, J=19.2Hz, 1H), 3.85(s, 2H), 2.70-1.60(m, 9H), 1.52(s, 3H), 1.20-1.00( m, 2H), 0.93 (s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）フェニル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｊ－３）の合成

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．８ｇ、１．６５ｍｍｏｌ）、ＨＡＴＵ（０．９４、２．４７ｍｍｏｌ）、ＤＩＰＥＡ（０．４２ｇ、３．３０ｍｍｏｌ）及びＤＭＦ（８ｍＬ）を、Ｎ_２下で室温で充填した。この溶液に、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（３－アミノベンジル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－Ｊ－２－Ｒ）（０．９３ｇ、１．６５ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水：６０：４０）によって精製し、薄黄色の固体として表題化合物を得た（０．４ｇ、２３．３１％）。ＬＣＭＳ：１０３４．８ ［Ｍ＋Ｈ］^＋。 S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ Synthesis of 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5-oxopentanoic acid (INX-J)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a , 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5 -Synthesis of tert-butyl oxopentanoate (INX-J-3)

procedure:
In a 50 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.8 g, 1.65 mmol), HATU (0.94, 2.47 mmol), DIPEA (0.42 g, 3.30 mmol) and DMF (8 mL) were mixed with N ₂ Filled at room temperature. Add (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-(3-aminobenzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl) to this solution. -6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1 ,2-d][1,3]dioxol-4-one (INX-J-2-R) (0.93 g, 1.65 mmol) was added at room temperature and stirred for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water: 60:40) to give the title compound as a pale yellow solid (0.4 g, 23.31%). LCMS: 1034.8 [M+H] ⁺ .

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）フェニル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｊ３）（０．４ｇ、０．３８ｍｍｏｌ）及びＴＨＦ（４ｍＬ）を充填した。この溶液に、ジエチルアミン（０．２７ｇ、３．８ｍｍｏｌ）を室温で添加し、３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、黄色の固体として表題化合物を得た（０．３ｇ、粗製）ＬＣＭＳ：８１２．５［Ｍ＋Ｈ］^＋． (S)-4-(2-aminoacetamide)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 Synthesis of tert-butyl':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5-oxopentanoate (INX-J-4)

procedure:
In a 50 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- tert-butyl)benzyl)phenyl)amino)-5-oxopentanoate (INX-J3) (0.4 g, 0.38 mmol) and THF (4 mL) were charged. Diethylamine (0.27 g, 3.8 mmol) was added to this solution at room temperature and stirred for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to give the title compound as a yellow solid (0.3 g, crude) LCMS: 812.5 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）フェニル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｊ－４）（０．３ｇ、０．３６ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、水（０．５ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０７５ｇ、０．７２ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０８８ｇ、０．４３ｍｍｏｌ）を室温で添加し、更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をジエチルエーテルによる粉砕によって精製し、淡黄色の固体として表題化合物を得た（０．２７ｇ、７８．２９％）。ＬＣＭＳ：９３２．５［Ｍ＋Ｈ］^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-tert-butyl-5-oxopentanoate (INX-J- 5) Synthesis

procedure:
In a 25 mL single-neck round bottom flask, (S)-4-(2-aminoacetamide)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- tert-butyl dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5-oxopentanoate (INX-J-4) (0.3 g, 0.36 mmol) and DCM (3 mL) were charged. To this solution was added Na ₂ CO ₃ (0.075 g, 0.72 mmol) dissolved in water (0.5 mL) followed by bromoacetyl bromide (0.088 g, 0.43 mmol) at room temperature, and an additional 1 Stir for hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was purified by trituration with diethyl ether to give the title compound as a pale yellow solid (0.27g, 78.29%). LCMS: 932.5 [M+H] ⁺

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）フェニル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｊ５）（０．２７ｇ、０．２８ｍｍｏｌ）を充填した。この溶液に、ＴＦＡ（０．１５ｇ、１．４０ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：Ｃ１８（２５０×２１．２）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝４８：５２）、保持時間１２．５分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．０２０ｇ、１０．６２％）。ＬＣＭＳ：Ｃ_４４Ｈ_５１ ^７９ＢｒＮ_３Ｏ_１１に対する計算値（８７６．２７）、実測値８７６．４０［Ｍ＋Ｈ］^＋；
^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４７－７．３６（ｍ， ５Ｈ）， ７．２３－７．２０（ｍ， ３Ｈ）， ６．９６（ｄ， １Ｈ）， ６．２７（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４５（ｓ， アセタール－Ｈ， １Ｈ）， ５．０６（ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．７０－４．２０（ｍ， ４Ｈ）， ４．１０－４．００（ｍ， ６Ｈ）， ２．７０－１．６０（ｍ， １３Ｈ）， １．５０（ｓ， ３Ｈ）， １．１０－１．０２（ｍ， ２Ｈ）， １．００（， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho Synthesis of [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5-oxopentanoic acid (INX-J)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((6aR,6bS,7S)) in DCM (2 mL). , 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a ,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl) tert-butyl amino)-5-oxopentanoate (INX-J5) (0.27 g, 0.28 mmol) was charged. To this solution was added TFA (0.15 g, 1.40 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: C18 (250 x 21.2) mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 48:52), retention time 12 .5 min) to give the title compound as an off-white solid (0.020 g, 10.62%). LCMS: Calculated value ^for _{C44H5179BrN3O11} ( _876.27 ), actual _value 876.40 [ _M +H] ⁺ ;
^1H NMR (400 MHz, MeOD): δ 7.47-7.36 (m, 5H), 7.23-7.20 (m, 3H), 6.96 (d, 1H), 6.27 ( d, J=10Hz, 1H), 6.04(s, 1H), 5.45(s, acetal-H, 1H), 5.06(d, J=5.2Hz, C16H, 1H), 4. 70-4.20 (m, 4H), 4.10-4.00 (m, 6H), 2.70-1.60 (m, 13H), 1.50 (s, 3H), 1.10- 1.02 (m, 2H), 1.00 (, 3H).

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）フェニル）アミノ）－５－オキソペンタン酸（ＩＮＸ－Ｊ）（０．２ｇ、０．２２ｍｍｏｌ）及びＤＭＦ（１ｍＬ）を充填した。この溶液に、Ｌ－システイン（０．０４１ｇ、０．３４ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＬＣＭＳによって示される反応の完了後、反応混合物を凍結乾燥させ、粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝７８：２２）、保持時間２０分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．０１０ｇ、４．７８％）。ＬＣＭＳ：Ｃ_４７Ｈ_５７ ^７９ＢｒＮ_４Ｏ_１３Ｓに対する計算値（９１７．３６）、実測値９１７．４［Ｍ＋Ｈ］^＋；
^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４７－７．３６（ｍ， ５Ｈ）， ７．２４－７．２０（ｍ， ３Ｈ）， ６．９６（ｄ， １Ｈ）， ６．２７（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４５（ｓ， アセタール－Ｈ， １Ｈ）， ５．０６（ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．７０－４．２０（ｍ， ４Ｈ）， ４．１０－３．９０（ｍ， ５Ｈ）， ３．５０－３．００（ｍ，４Ｈ）， ２．７０－１．６０（ｍ， １３Ｈ）， １．５０（ｓ， ３Ｈ）， １．１０－１．０２（ｍ， ２Ｈ）， １．００（， ３Ｈ）。 (S)-4-(2-(2-(((R)-2-amino-2-carboxyethyl)thio)acetamido)acetamido)-5-((3-(4-((6aR,6bS,7S , 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a ,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl) Synthesis of amino)-5-oxopentanoic acid (INX-J-CYS)

procedure:
In a 10 mL single neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R) , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-5-oxo Pentanoic acid (INX-J) (0.2 g, 0.22 mmol) and DMF (1 mL) were charged. To this solution was added L-cysteine (0.041 g, 0.34 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by LCMS, the reaction mixture was lyophilized and the crude was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = in water. Purification by 0.05% TFA, B=acetonitrile, A:B=78:22), retention time 20 min) gave the title compound as an off-white solid (0.010 g, 4.78%). LCMS: Calculated value ^for _{C47H5779BrN4O13S} ( _917.36 ), actual _value 917.4 [ _M +H] ⁺ ;
^1H NMR (400 MHz, MeOD): δ 7.47-7.36 (m, 5H), 7.24-7.20 (m, 3H), 6.96 (d, 1H), 6.27 ( d, J=10Hz, 1H), 6.04(s, 1H), 5.45(s, acetal-H, 1H), 5.06(d, J=5.2Hz, C16H, 1H), 4. 70-4.20 (m, 4H), 4.10-3.90 (m, 5H), 3.50-3.00 (m, 4H), 2.70-1.60 (m, 13H), 1.50 (s, 3H), 1.10-1.02 (m, 2H), 1.00 (, 3H).

（６Ｓ，８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－６，９－ジフルオロ－１１，１６，１７－トリヒドロキシ１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（ＩＮＸ－ＳＭ－２５－１）の合成

手順：
３５ｍＬのガラスバイアルに、フルオシノロンアセトニド（１ｇ、２．２３ｍｍｏｌ）及びフルオロホウ酸（１０ｍＬ）を充填した。混合物を室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、結果として得られた固体を濾過し、真空下で乾燥させて、白色の固体として表題化合物を得た（０．８ｇ、８７．７７％）。ＬＣＭＳ：４１３．２［Ｍ＋Ｈ］^＋。 (2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)methyl)phenyl)-2, 6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H -Synthesis of naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-25)

(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy17-(2-hydroxyacetyl)-10,13-dimethyl-6 , 7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (INX-SM-25-1)

procedure:
A 35 mL glass vial was charged with fluocinolone acetonide (1 g, 2.23 mmol) and fluoroboric acid (10 mL). The mixture was stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the resulting solid was filtered and dried under vacuum to give the title compound as a white solid (0.8 g, 87.77 %). LCMS: 413.2 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（６－（４－ホルミルベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３２－４）（０．１０ｇ、０．３３ｍｍｏｌ）、（６Ｓ，８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－６，９－ジフルオロ－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（ＩＮＸ－ＳＭ－２５－１）（０．１ｇ、０．２４ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、ＭｇＳＯ_４（０．１８ｇ、１．５９ｍｍｏｌ）及びＨＣｌＯ_４（０．１５ｇ、１．５９ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝７０：３０）、保持時間９．９８分）によって精製し、白色の固体として表題化合物を得た（０．０２４ｇ、１３．４０％）。ＬＣＭＳ：６２４．４［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ，）： δ：８．５５（ｂｒ ｓ， １Ｈ）， ７．３６－７．３３（ｍ， ３Ｈ）， ７．１６（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．３６－６．３３（ｍ， ２Ｈ）， ５．６６－５．５１（ｍ， Ｊ＝４５．６Ｈｚ， ＣＨＦ， １Ｈ） ５．４８（ｓ， アセタール－Ｈ， １Ｈ）， ５．０７（ｄ， Ｊ＝３．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ５．０７－５．０６（ｍ， １Ｈ）， ４．６３（ｄ， Ｊ＝１９．２Ｈｚ， １Ｈ）， ４．３６－４．３１（ｍ， ２Ｈ）， ３．５８－３．５４（ｍ， １Ｈ）， ２．７５－１．６０（ｍ， １８Ｈ）， １．６０（ｓ， ３Ｈ）， １．００（ｓ， ３Ｈ）。 (2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)methyl)phenyl)-2, 6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H -Synthesis of naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-25)

procedure:
In a 25 mL single-neck round bottom flask, add tert-butyl (6-(4-formylbenzyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-32-4) (0.10 g, 0.33 mmol), (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10 ,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (INX-SM-25-1 ) (0.1 g, 0.24 mmol) and DCM (3 mL) were charged. To this solution were added MgSO ₄ (0.18 g, 1.59 mmol) and HClO ₄ (0.15 g, 1.59 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 70:30), retention time 9. 98 min) to give the title compound as a white solid (0.024 g, 13.40%). LCMS: 624.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD,): δ: 8.55 (br s, 1H), 7.36-7.33 (m, 3H), 7.16 ( d, J=8.0Hz, 2H), 6.36-6.33(m, 2H), 5.66-5.51(m, J=45.6Hz, CHF, 1H) 5.48(s, Acetal-H, 1H), 5.07 (d, J=3.2Hz, C16H, 1H), 5.07-5.06 (m, 1H), 4.63 (d, J=19.2Hz, 1H) ), 4.36-4.31 (m, 2H), 3.58-3.54 (m, 1H), 2.75-1.60 (m, 18H), 1.60 (s, 3H), 1.00 (s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２３－１）の合成

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．３８ｇ、０．７８ｍｍｏｌ）、ＨＡＴＵ（０．６０８ｇ、１．６ｍｍｏｌ）、及びＤＭＦ（５ｍＬ）を充填した。この溶液に、ＤＩＰＥＡ（０．２５ｇ、２．０ｍｍｏｌ）、続いて、（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）メチル）フェニル）－２，６ｂ－ジフルオロ－７－ヒドロキ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－２５）（０．５ｇ、０．８０ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水：５０：５０）によって精製し、薄黄色の固体として表題化合物を得た（０．３ｇ、３４．３９％）。ＬＣＭＳ １０８８．６［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a , 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane-2- Synthesis of amino)-5-oxopentanoic acid (INX-A23)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS,6bR,7S,8aS , 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7 , 8,8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl) Synthesis of tert-butyl)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A23-1)

procedure:
In a 10 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.38 g, 0.78 mmol), HATU (0.608 g, 1.6 mmol), and DMF (5 mL) were charged. To this solution was added DIPEA (0.25 g, 2.0 mmol) followed by (2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro [3.3]Heptan-2-yl)methyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7 ,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one ( INX-SM-25) (0.5 g, 0.80 mmol) was added at room temperature and stirred for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water: 50:50) to give the title compound as a pale yellow solid (0.3 g, 34.39%). LCMS 1088.6 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２３－１）（０．１２ｇ、０．１１ｍｍｏｌ）及びＴＨＦ（２ｍＬ）を充填した。この溶液に、ジエチルアミン（０．０８ｇ、１．１０ｍｍｏｌ）を室温で添加し、３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテルで粉砕して、黄色の固体として表題化合物を得た（０．０８ｇ、８３．７８％）ＬＣＭＳ：８６６．６ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-aminoacetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)- Synthesis of tert-butyl 5-oxopentanoate (INX-A23-2)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-( (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ tert-butyl 1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A23-1) (0.12 g, 0.11 mmol ) and THF (2 mL). Diethylamine (0.08 g, 1.10 mmol) was added to this solution at room temperature and stirred for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether to give the title compound as a yellow solid (0.08 g, 83.78%) LCMS: 866.6 [ M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２３－２）（０．０８ｇ、０．０９ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、水（１ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０３９ｇ、０．３６ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０３７ｇ、０．１８ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、５０：５０）によって精製し、薄黄色として表題化合物を得た（０．０８０ｇ、４０％）。ＬＣＭＳ：９８６．６ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a , 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane-2- Synthesis of tert-butyl)amino)-5-oxopentanoate (INX-A23-3)

procedure:
In a 10 mL single neck round bottom flask, (S)-4-(2-aminoacetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3 ] tert-Butylheptan-2-yl)amino)-5-oxopentanoate (INX-A23-2) (0.08 g, 0.09 mmol) and DCM (3 mL) were charged. To this solution was added Na ₂ CO ₃ (0.039 g, 0.36 mmol) dissolved in water (1 mL) followed by bromoacetyl bromide (0.037 g, 0.18 mmol) and stirred at room temperature for 1 h. . After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 50:50) to give the title compound as a pale yellow color (0.080 g, 40%). LCMS: 986.6 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２３－３）（０．０８０ｇ、０．０８ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．０４８ｇ、０．４５ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝５８：４２）、保持時間１４．６２分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．００８ｇ、１０．６０％）。ＬＣＭＳ：Ｃ_４５Ｈ_５４ ^７９ＢｒＦ_２Ｎ_３Ｏ_１１Ｎａに対する計算値（９５３．８０）、実測値９５３．５［Ｍ＋Ｎａ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ， 主要プロトン割り当て）： δ：７．３６－７．３４（ｍ， ３Ｈ）， ７．１７（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．３８－６．３５（ｍ， ２Ｈ）， ５．６６－５．４９（ｍ， ＣＨＦ， １Ｈ）， ５．４８（ｓ， １Ｈ， アセタール－Ｈ）， ５．０７（ｄ， Ｊ＝４．４ Ｈｚ， １Ｈ， Ｃ１６Ｈ）， ４．６４（ｄ， １Ｈ）， ４．３７－４．３２（ｍ， ２Ｈ）， ４．１５－４．１０（ｍ， １Ｈ）， ３．９４－３．９０（ｍ， ４Ｈ）， ２．６８（ｄ， ２Ｈ）， ２．５０－１．６５（ｍ，２２Ｈ）， １．６０（ｓ， ３Ｈ）， ０．９０（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a , 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane-2- Synthesis of amino)-5-oxopentanoic acid (INX-A23)

procedure:
In a 10 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS) , 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8 , 8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl) Tert-butyl spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A23-3) (0.080 g, 0.08 mmol) and DCM (2 mL) were charged. To this solution was added TFA (0.048 g, 0.45 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 58:42), retention time 14. 62 min) to give the title compound as an off-white solid (0.008 g, 10.60%). LCMS: Calculated value for C ₄₅ H ₅₄ ⁷⁹ BrF ₂ N ₃ O ₁₁ Na (953.80), actual value 953.5 [M+Na] ⁺ ; ¹ H NMR (400 MHz, MeOD, major proton assignment): δ: 7 .36-7.34 (m, 3H), 7.17 (d, J=8.0Hz, 2H), 6.38-6.35 (m, 2H), 5.66-5.49 (m, CHF, 1H), 5.48 (s, 1H, acetal-H), 5.07 (d, J=4.4 Hz, 1H, C16H), 4.64 (d, 1H), 4.37-4 .32 (m, 2H), 4.15-4.10 (m, 1H), 3.94-3.90 (m, 4H), 2.68 (d, 2H), 2.50-1.65 (m, 22H), 1.60 (s, 3H), 0.90 (s, 3H).

（（Ｓ）－５－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－６－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－６－オキソヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ Ａ６－１）の合成

手順：
２５ｍＬの一つ口丸底フラスコに、Ｎ２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシル）－Ｎ６－（ｔｅｒｔ－ブトキシカルボニル）－Ｌ－リジン（ＩＮＸ－Ｗ－２）（０．２５ｇ、０．４８ｍｍｏｌ）、ＨＡＴＵ（０．２７ｇ、０．７２ｍｍｏｌ）、及びＤＭＦ（３ｍＬ）を充填した。この溶液に、ＤＩＰＥＡ（０．１２ｇ、０．９６ｍｍｏｌ）及び（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）メチル）フェニル）－２，６ｂ－ジフルオロ－７－ヒドロキ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－２５）（０．３ｇ、０．４８ｍｍｏｌ）を反応物に添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、５０：５０）によって精製し、薄黄色の固体として表題化合物を得た（０．２８ｇ、５１．４６％）。ＬＣＭＳ：１１３１．７［Ｍ＋Ｈ］^＋。 (S)-6-amino-2-(2-(2-bromoacetamido)acetamide)-N-(6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a, 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane Synthesis of -2-yl)hexanamide (INX-A6)

((S)-5-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-6-((6-(4-((2S,6aS,6bR,7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b, 7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl ) Synthesis of tert-butyl)benzyl)spiro[3.3]heptan-2-yl)amino)-6-oxohexyl)carbamate (INX A6-1)

procedure:
In a 25 mL single-necked round bottom flask, add N2-((((9H-fluoren-9-yl)methoxy)carbonyl)glycyl)-N6-(tert-butoxycarbonyl)-L-lysine (INX-W-2). (0.25 g, 0.48 mmol), HATU (0.27 g, 0.72 mmol), and DMF (3 mL). To this solution was added DIPEA (0.12 g, 0.96 mmol) and (2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3. 3] heptan-2-yl)methyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8, 8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM -25) (0.3 g, 0.48 mmol) was added to the reaction and stirred for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 50:50) to give the title compound as a pale yellow solid (0.28g, 51.46%). LCMS: 1131.7 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（（Ｓ）－５－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－６－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－６－オキソヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ Ａ６－１）（０．２５ｇ、０．２２ｍｍｏｌ）及びＴＨＦ（３ｍＬ）を充填した。この溶液に、ジエチルアミン（０．１６ｇ、２．０ｍｍｏｌ）を室温で添加し、３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテルで粉砕して、黄色の固体として表題化合物を得た（０．１９ｇ、９４．２１％）。ＬＣＭＳ：９０９．５［Ｍ＋Ｈ］^＋。 ((S)-5-(2-aminoacetamide)-6-((6-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b- Difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)-6 Synthesis of tert-butyl -oxyhexyl)carbamate (INX A6-2)

procedure:
In a 25 mL single neck round bottom flask, add ((S)-5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-6-((6-(4- ((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4- Oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d] tert-Butyl [1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-6-oxohexyl)carbamate (INX A6-1) (0.25g, 0 .22 mmol) and THF (3 mL). Diethylamine (0.16 g, 2.0 mmol) was added to this solution at room temperature and stirred for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether to give the title compound as a yellow solid (0.19 g, 94.21%). LCMS: 909.5 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（（Ｓ）－５－（２－アミノアセトアミド）－６－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）－６－オキシヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ Ａ６－２）（０．１９ｇ、０．２１ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、水（１ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０９３ｇ、０．８３ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０８８ｇ、０．４１ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、５０：５０）によって精製し、薄黄色の固体として表題化合物を得た（０．０９０ｇ、４１．８１％）。ＬＣＭＳ：１０２９．７［Ｍ＋Ｈ］^＋。 ((S)-5-(2-(2-bromoacetamide)acetamide)-6-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12, 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane-2 Synthesis of tert-butyl-yl)amino)-6-oxohexyl)carbamate (INX A6-3)

procedure:
In a 10 mL single-neck round bottom flask, add ((S)-5-(2-aminoacetamide)-6-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR) , 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b , 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3. 3] tert-Butyl heptan-2-yl)-6-oxyhexyl)carbamate (INX A6-2) (0.19 g, 0.21 mmol) and DCM (3 mL) were charged. To this solution was charged water (1 mL). ) was added, followed by _{bromoacetyl bromide} (0.088 g, 0.41 mmol) and stirred at room temperature for 1 h. After completion, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO and evaporated _under vacuum. The crude was purified by reverse phase column chromatography (acetonitrile:water, 50:50) to give the title compound as a pale yellow solid (0.090 g, 41.81%). LCMS: 1029.7 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＣＭ（４ｍＬ）中の（（Ｓ）－５－（２－（２－ブロモアセトアミド）アセトアミド）－６－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－６－オキソヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ Ａ６－３）（０．０７０ｇ、０．０６７ｍｍｏｌ）を充填した。この溶液に、ＴＦＡ（０．０３８ｇ、０．３３ｍｍｏｌ）を室温で添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝５０：５０）、保持時間１６分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．００５ｇ、７．０５％）。ＬＣＭＳ：９２９．５９［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．３６－７．３４（ｍ， ３Ｈ）， ７．１６（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．３８－６．３４（ｍ， ２Ｈ）， ５．６７－５．４９（ｍ， ＣＨＦ， Ｊ＝４８．８Ｈｚ， １Ｈ） ５．４９（ｓ， アセタール－Ｈ， １Ｈ）， ５．０８（ｄ， Ｊ＝３．６Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．６５（ｄ， Ｊ＝１９．６Ｈｚ， １Ｈ）， ４．３７－４．３２（ｍ， ３Ｈ）， ４．２０－４．１０（ｍ， ２Ｈ）， ３．９７（ｓ， ２Ｈ）， ３．９４（ｄ， ２Ｈ）， ３．００－２．９０（ｍ， ２Ｈ）， ２．８０－２．６０（ｍ， ２Ｈ）， ２．５０－１．５５（ｍ， ２０Ｈ）， １．６０（ｓ， ３Ｈ）， １．５５－１．４０（ｍ， ２Ｈ）， １．００（ｓ， ３Ｈ）。 (S)-6-amino-2-(2-(2-bromoacetamido)acetamide)-N-(6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a, 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane Synthesis of -2-yl)hexanamide 2,2,2-trifluoroacetate (INX-A6)

procedure:
In a 10 mL single neck round bottom flask, add ((S)-5-(2-(2-bromoacetamido)acetamide)-6-((6-(4-((2S,6aS, 6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4, 6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole Loaded with tert-butyl-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-6-oxohexyl)carbamate (INX A6-3) (0.070 g, 0.067 mmol) . To this solution was added TFA (0.038 g, 0.33 mmol) at room temperature and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 50: 50), retention time 16 min) to give the title compound as an off-white solid (0.005 g, 7.05%). LCMS: 929.59 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ 7.36-7.34 (m, 3H), 7.16 (d, J=8.0Hz, 2H), 6 .38-6.34 (m, 2H), 5.67-5.49 (m, CHF, J=48.8Hz, 1H) 5.49 (s, acetal-H, 1H), 5.08 (d , J=3.6Hz, C16H, 1H), 4.65(d, J=19.6Hz, 1H), 4.37-4.32(m, 3H), 4.20-4.10(m, 2H), 3.97 (s, 2H), 3.94 (d, 2H), 3.00-2.90 (m, 2H), 2.80-2.60 (m, 2H), 2.50 -1.55 (m, 20H), 1.60 (s, 3H), 1.55-1.40 (m, 2H), 1.00 (s, 3H).

（２－（（（Ｓ）－４－アミノ－１－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－１，４－ジオキソブタン－２－イル）アミノ）－２－オキソエチル）カルバミン酸（９Ｈ－フルオレン－９－イル）メチル（ＩＮＸ－Ａ１１－１）の合成

手順：
２５ｍＬの一つ口丸底フラスコに、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－３２）（０．５０ｇ、０．８５ｍｍｏｌ）、（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）グリシル－Ｌ－アスパラギン（ＩＮＸ－Ｘ－２）（０．５２ｇ、１．２７ｍｍｏｌ）及びＤＭＦ（５ｍＬ）を充填した。この溶液に、ＨＡＴＵ（０．６４ｇ、１．７０ｍｍｏｌ）及びＤＩＰＥＡ（０．３２ｇ、２．５５ｍｍｏｌ）を室温で添加し、１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、５０：５０）によって精製し、薄黄色の固体として表題化合物を得た（０．３２ｇ、３８．３４％）。ＬＣＭＳ：９８１．５［Ｍ＋Ｈ］^＋。 (S)-2-(2-(2-bromoacetamide)acetamide)-N1-(6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)succinamide (INX-A11) synthesis

(2-(((S)-4-amino-1-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-1,4-dioxobutan-2- Synthesis of (9H-fluoren-9-yl)methyl)amino)-2-oxoethyl)carbamate (INX-A11-1)

procedure:
In a 25 mL single neck round bottom flask, add (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl) ) methyl) phenyl) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-32) (0.50 g, 0.85 mmol), (((9H-Fluoren-9-yl)methoxy)carbonyl)glycyl-L-asparagine (INX-X-2) (0.52 g, 1.27 mmol) and DMF (5 mL) were charged. To this solution were added HATU (0.64 g, 1.70 mmol) and DIPEA (0.32 g, 2.55 mmol) at room temperature and stirred for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 50:50) to give the title compound as a pale yellow solid (0.32g, 38.34%). LCMS: 981.5 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（２－（（（Ｓ）－４－アミノ－１－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－１，４－ジオキソブタン－２－イル）アミノ）－２－オキソエチル）カルバミン酸（９Ｈ－フルオレン－９－イル）メチル（ＩＮＸ－Ａ１１－１）（０．３２ｇ、０．３２ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ジエチルアミン（０．２３ｇ、３．２３ｍｍｏｌ）を室温で添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテルで粉砕して、黄色の固体として表題化合物を得た（０．２０ｇ、８２．３５％）。ＬＣＭＳ：７５９．５［Ｍ＋Ｈ］^＋。 (S)-2-(2-aminoacetamide)-N1-(6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2 -hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)succinamide (INX-A11-2)

procedure:
In a 10 mL single neck round bottom flask, add (2-(((S)-4-amino-1-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino )-1,4-dioxobutan-2-yl)amino)-2-oxoethyl)carbamic acid (9H-fluoren-9-yl)methyl (INX-A11-1) (0.32 g, 0.32 mmol) and THF ( 5 mL). Diethylamine (0.23 g, 3.23 mmol) was added to this solution at room temperature and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether to give the title compound as a yellow solid (0.20 g, 82.35%). LCMS: 759.5 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－アミノアセトアミド）－Ｎ１－（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）スクシンアミド（ＩＮＸ－Ａ１１－２）（０．１ｇ、０．１３ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、水（０．５ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０５５ｇ、０．５２ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０５３ｇ、０．２６ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製の表題化合物を分取ＨＰＬＣ
（カラム：ＹＭＣ Ｃ１８ １２０ｇｍ、５０μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ、５５：４５）、保持時間１５．５１分）によって精製し、白色の固体として表題化合物を得た（０．０３５ｇ、３０．５％）。ＬＣＭＳ：Ｃ_４４Ｈ_５６ ^７９ＢｒＦＮ４Ｏ_１０に対する計算値（８７９．３２）、実測値８７９．４［Ｍ＋Ｈ］^＋； (S)-2-(2-(2-bromoacetamide)acetamide)-N1-(6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)succinamide (INX-A11) synthesis

procedure:
In a 25 mL single neck round bottom flask, (S)-2-(2-aminoacetamide)-N1-(6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) )-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)succinamide ( INX-A11-2) (0.1 g, 0.13 mmol) and DCM (3 mL) were charged. To this solution was added Na ₂ CO ₃ (0.055 g, 0.52 mmol) dissolved in water (0.5 mL) followed by bromoacetyl bromide (0.053 g, 0.26 mmol) at room temperature for 1 h. Stirred. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude title compound was analyzed by preparative HPLC.
(Column: YMC C18 120 gm, 50 μm, Mobile phase: A = 0.05% TFA in water, B = Acetonitrile, A:B, 55:45), retention time 15.51 min) as a white solid. The title compound was obtained (0.035g, 30.5%). LCMS: Calculated value for C ₄₄ H ₅₆ ⁷⁹ BrFN4O ₁₀ (879.32), actual value 879.4 [M+H] ⁺ ;

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（２－ブロモアセトアミド）アセトアミド）－Ｎ１－（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）スクシンアミド（ＩＮＸ－Ａ１１）（０．１ｇ、０．１１ｍｍｏｌ）及びＤＭＦ（１ｍＬ）を充填した。この溶液に、Ｌ－システイン（０．０２６ｇ、０．２２ｍｍｏｌ）を添加し、室温で１６時間撹拌した。ＬＣＭＳによって示される反応の完了後、反応混合物を凍結乾燥させ、粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル：ＭＴＢＥ、９０：１０、Ａ：Ｂ＝６０：４０）、保持時間１６分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．００９６ｇ、９．６１％）。ＬＣＭＳ：９２０．５［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）：８．５４（ｔ， １Ｈ）， ８．３４（ｄ， １Ｈ）， ７．８８（ｂｒ ｄ， ３Ｈ）， ７．４３（ｂｒ ｓ， １Ｈ）， ７．３５－７．３１（ｍ， ３Ｈ）， ７．１５（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．８７（ｂｒ ｓ， １Ｈ）， ６．１７（ｄ， Ｊ＝１０Ｈｚ １Ｈ）， ５．９４（ｓ， １Ｈ）， ５．３９（ｓ， アセタール－Ｈ， １Ｈ）， ５．１０（ｂｒ ｓ， １Ｈ）， ４．９３（ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．８０（ｂｒ ｓ， １Ｈ）， ４．５３－４．４３（ｍ， ２Ｈ）， ４．３０－４．１６（ｍ， ２Ｈ）， ４．０１－３．９８（ｍ， １Ｈ）， ３．７１（ｄ， ２Ｈ）， ２．９０－３．００（ｍ， ２Ｈ）， ２．７０－１．６０（ｍ， ２５Ｈ）， １．４０（ｓ， ３Ｈ）， １．０２－０．９５（ｍ， ２Ｈ）， ０．８７（ｓ， ３Ｈ）。 S-(2-((2-(((S)-4-amino-1-((6-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a ,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3] Synthesis of L-cysteine compound (INX-A11-CYS)

procedure:
In a 10 mL single-neck round bottom flask, (S)-2-(2-(2-bromoacetamido)acetamide)-N1-(6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12, 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane-2 -yl)succinamide (INX-A11) (0.1 g, 0.11 mmol) and DMF (1 mL) were charged. To this solution was added L-cysteine (0.026 g, 0.22 mmol) and stirred at room temperature for 16 hours. After completion of the reaction as indicated by LCMS, the reaction mixture was lyophilized and the crude was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.05% TFA in water, B =acetonitrile:MTBE, 90:10, A:B=60:40), retention time 16 min) to give the title compound as an off-white solid (0.0096 g, 9.61%). LCMS: 920.5 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): 8.54 (t, 1H), 8.34 (d, 1H), 7.88 (br d, 3H), 7. 43 (br s, 1H), 7.35-7.31 (m, 3H), 7.15 (d, J=8.0Hz, 2H), 6.87 (br s, 1H), 6.17 ( d, J=10Hz 1H), 5.94(s, 1H), 5.39(s, acetal-H, 1H), 5.10(br s, 1H), 4.93(d, J=5. 2Hz, C16H, 1H), 4.80 (br s, 1H), 4.53-4.43 (m, 2H), 4.30-4.16 (m, 2H), 4.01-3.98 (m, 1H), 3.71 (d, 2H), 2.90-3.00 (m, 2H), 2.70-1.60 (m, 25H), 1.40 (s, 3H), 1.02-0.95 (m, 2H), 0.87 (s, 3H).

（Ｅ）－（３－（（２－トシルヒドラゾノ）メチル）スピロ［３．３］ヘプタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４５－１）の合成

手順：
５０ｍＬの一つ口丸底フラスコに、窒素下で（３－ホルミルスピロ［３．３］ヘプタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（１．０ｇ、４．１８ｍｍｏｌ）及びＥｔＯＨ（２５ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホンヒドラジド（０．９３４ｇ、６．２７６ｍｍｏｌ）を添加し、触媒量のＡｃＯＨ（０．２ｍＬ）を添加し、室温で０．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、固体を形成し、それを濾過した。化合物を真空下で乾燥させ、白色の固体として表題化合物を得た（０．８１ｇ、４６．９４％）。ＬＣＭＳ：４０７．５３［Ｍ＋Ｈ］^＋ (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminospiro[3.3]heptan-1-yl)methyl)phenyl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-45)

(E) Synthesis of tert-butyl-(3-((2-tosylhydrazono)methyl)spiro[3.3]heptan-1-yl)carbamate (INX-SM-45-1)

procedure:
A 50 mL single neck round bottom flask was charged with tert-butyl (3-formylspiro[3.3]heptan-1-yl)carbamate (1.0 g, 4.18 mmol) and EtOH (25 mL) under nitrogen. did. To this solution was added p-toluenesulfone hydrazide (0.934 g, 6.276 mmol) and a catalytic amount of AcOH (0.2 mL) and stirred at room temperature for 0.5 h. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water to form a solid, which was filtered. The compound was dried under vacuum to give the title compound as a white solid (0.81 g, 46.94%). LCMS: 407.53 [M+H] ⁺

手順：
窒素下で（Ｅ）－（３－（（２－トシルヒドラゾノ）メチル）スピロ［３．３］ヘプタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４５－１）（０．５ｇ、１．２２ｍｍｏｌ）及びジオキサン（２０ｍＬ）を含む３５ｍＬのバイアル。この溶液に、（４－ホルミルフェニル）ボロン酸（０．５５１ｇ、３．６８ｍｍｏｌ）及びＫ_２ＣＯ_３（０．５０８ｇ、３．６８ｍｍｏｌ）を室温で添加し、１００℃で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、３０：７０）によって精製し、黄色の粘着性固体として表題化合物を得た（０．１８０ｇ、４４．７％）。ＬＣＭＳ：３３０［Ｍ＋Ｈ］^＋。 Synthesis of tert-butyl (3-(4-formylbenzyl)spiro[3.3]heptan-1-yl)carbamate (INX-SM-45-2)

procedure:
tert-butyl (E)-(3-((2-tosylhydrazono)methyl)spiro[3.3]heptan-1-yl)carbamate (INX-SM-45-1) (0.5 g, 1 .22 mmol) and dioxane (20 mL). To this solution was added (4-formylphenyl)boronic acid (0.551 g, 3.68 mmol) and K ₂ CO ₃ (0.508 g, 3.68 mmol) at room temperature and stirred at 100° C. for an additional 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude material was purified by silica gel column chromatography (ethyl acetate/hexanes, 30:70) to give the title compound as a yellow sticky solid (0.180 g, 44.7%). LCMS: 330 [M+H] ⁺ .

手順：
５０ｍＬの一つ口丸底フラスコに、（３－（４－ホルミルベンジル）スピロ［３．３］ヘプタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４５－２）（０．４００ｇ、１．２４４ｍｍｏｌ）及び（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．４５７ｇ、１．２４４ｍｍｏｌ）を充填した。これをＤＣＭ（８ｍＬ）に溶解させ、ＭｇＳＯ_４（０．７３０ｇ、６．０７ｍｍｏｌ）を溶液中に添加した。この溶液に、ＨＣｌＯ_４（０．４０５ｇ、６．０７ｍｍｏｌ）を添加し、室温で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液によってクエンチし、真空上で濃縮した。粗製物を冷水で粉砕し、参加したものを濾過し、真空下で乾燥させて、粗製物を得た。粗製物の一部（０．１０ｇ）を分取ＨＰＬＣ（カラム：Ｃ１８（２５０×２１．２）ＭＭ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝５３：４６、保持時間１２分）によって精製し、表題化合物を得た（０．０１３ｇ、７％）。ＬＣＭＳ：５８８．４［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．４０（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．２８（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０ ＆ １．６Ｈｚ １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４９（ｓ， アセタール－Ｈ， １Ｈ）， ５．０８（ｄ， Ｊ＝４．８Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．６１（ｄ， Ｊ＝１９．２Ｈｚ， １Ｈ）， ４．４４－４．４３（ｍ， １Ｈ）， ４．３４（ｄ， Ｊ＝１９．２Ｈｚ， １Ｈ）， ３．０５－２．９５（ｍ， １Ｈ）， ２．８０－１．６０（ｍ， ２０Ｈ）， １．５２（ｓ， ３Ｈ）， １．２０－１．００（ｍ， ２Ｈ）， １．０１（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminospiro[3.3]heptan-1-yl)methyl)phenyl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-45)

procedure:
In a 50 mL single-neck round bottom flask, add tert-butyl (3-(4-formylbenzyl)spiro[3.3]heptan-1-yl)carbamate (INX-SM-45-2) (0.400 g, 1.244 mmol) and (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7 , 8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0.457g, 1.244mmol ) was filled. This was dissolved in DCM (8 mL) and MgSO ₄ (0.730 g, 6.07 mmol) was added into the solution. To this solution was added HClO ₄ (0.405 g, 6.07 mmol) and stirred for an additional 2 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched by saturated _NaHCO3 solution and concentrated on vacuum. The crude material was triturated with cold water, the ingredients were filtered and dried under vacuum to obtain the crude material. A portion of the crude material (0.10 g) was subjected to preparative HPLC (column: C18 (250 x 21.2) MM, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A: B = 53:46, retention time 12 min) to give the title compound (0.013 g, 7%). LCMS: 588.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ 7.47 (d, J = 10.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H) , 7.28 (d, J=8.0Hz, 2H), 6.27 (dd, J=10.0 & 1.6Hz 1H), 6.04 (s, 1H), 5.49 (s, acetal -H, 1H), 5.08 (d, J=4.8Hz, C16H, 1H), 4.61 (d, J=19.2Hz, 1H), 4.44-4.43 (m, 1H) , 4.34 (d, J=19.2Hz, 1H), 3.05-2.95 (m, 1H), 2.80-1.60 (m, 20H), 1.52 (s, 3H) , 1.20-1.00 (m, 2H), 1.01 (s, 3H).

（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ８－１）の合成

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．４１０ｇ、０．８５０ｍｍｏｌ）を充填し、ＤＭＦ（５ｍＬ）中のＨＡＴＵ（０．４８５ｇ、１．２７ｍｍｏｌ）をＤＩＰＥＡ（０．３２９ｇ、２．５５ｍｍｏｌ）に室温で添加した。この溶液に、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノスピロ［３．３］ヘプタン－１－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－４５）（０．５ｇ、０．８５０ｍｍｏｌ）を室温で添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物を正常相カラムクロマトグラフィー（ＤＣＭ：ＭｅＯＨ、９０：１０）によって精製し、薄黄色の固体として表題化合物を得た（０．３ｇ、３３．５１％）。ＬＣＭＳ：１０５２．９［Ｍ＋Ｈ］^＋ (4S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-1-yl)amino)-5-oxo Synthesis of pentanoic acid (INX-A8)

(4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a ,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3] Synthesis of tert-butyl heptane-1-yl)amino)-5-oxopentanoate (INX-A8-1)

procedure:
In a 25 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.410 g, 0.850 mmol) was charged with HATU (0.485 g, 1.27 mmol) in DMF (5 mL) with DIPEA (0.329 g, 2.55 mmol). was added at room temperature. Add (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3-aminospiro[3.3]heptan-1-yl)methyl)phenyl)- to this solution. 7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2 ',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-45) (0.5 g, 0.850 mmol) was added at room temperature; The mixture was stirred for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude material was purified by normal phase column chromatography (DCM:MeOH, 90:10) to give the title compound as a pale yellow solid (0.3 g, 33.51%). LCMS: 1052.9 [M+H] ⁺

手順：
２５ｍＬの一つ口丸底フラスコに、ＴＨＦ（５ｍＬ）中の（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ８－１）（０．３ｇ、０．２８５ｍｍｏｌ）を充填した。この溶液に、ジエチルアミン（０．２０８ｇ、２．８５ｍｍｏｌ）を室温で添加し、室温で２．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空上で直接濃縮して粗製物を得て、ＤＣＭ及びヘキサンによる粉砕によって精製し、真空下で乾燥させて、白色の固体として表題化合物を得た（０．１７５ｇ、７４％）。ＬＣＭＳ：８３１ ［Ｍ＋Ｈ］^＋。 (4S)-4-(2-aminoacetamide)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-1-yl)amino)-tert-butyl-5-oxopentanoate Synthesis of (INX-A8-2)

procedure:
In a 25 mL single neck round bottom flask, add (4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(( 3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2 ,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1, 3] Tert-butyl dioxol-10-yl)benzyl)spiro[3.3]heptan-1-yl)amino)-5-oxopentanoate (INX-A8-1) (0.3 g, 0.285 mmol) Filled. To this solution was added diethylamine (0.208 g, 2.85 mmol) at room temperature and stirred at room temperature for 2.5 hours. After completion of the reaction as indicated by TLC, the reaction mixture was directly concentrated in vacuo to give the crude product, which was purified by trituration with DCM and hexanes and dried under vacuum to give the title compound as a white solid. (0.175g, 74%). LCMS: 831 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、ＤＣＭ（５ｍＬ）中（４Ｓ）－４－（２－アミノアセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ８－２）（０．１７５ｇ、０．２１０ｍｍｏｌ）を充填した。この溶液に、水（１ｍｌ）に溶解した室温のＮａ_２ＣＯ_３（０．０４４ｇ、０．４２１ｍｍｏｌ）を添加し、次いで、これにブロモアセチルブロミド（０．０４２ｇ、０．２１０ｍｍｏｌ）を室温で添加し、１．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＭＤＣで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、５０：５０）によって精製し、オフホワイト色の固体として表題化合物を得た（０．０５５ｇ、２７．４３％）。ＬＣＭＳ：Ｃ_４９Ｈ_６５ ^７９ＢｒＦＮ_３Ｏ_１１に対する計算値（９５０．３８）、実測値９５０．６［Ｍ＋Ｈ］^＋。 (4S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-1-yl)amino)-5-oxo Synthesis of tert-butyl pentanoate (INX-A8-3)

procedure:
In a 25 mL single neck round bottom flask, add (4S)-4-(2-aminoacetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R) in DCM (5 mL). , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane- tert-Butyl (1-yl)amino)-5-oxopentanoate (INX-A8-2) (0.175g, 0.210mmol) was charged. To this solution was added Na ₂ CO ₃ (0.044 g, 0.421 mmol) dissolved in water (1 ml) at room temperature, to which was then added bromoacetyl bromide (0.042 g, 0.210 mmol) at room temperature. and stirred for 1.5 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with MDC. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 50:50) to give the title compound as an off-white solid (0.055g, 27.43%). LCMS: Calcd ^for _{C49H6579BrFN3O11} ( _950.38 ), found _950.6 [ _M +H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ８－３）（０．２５０ｇ、０．２６２ｍｍｏｌ）及びＤＣＭ（１５ｍＬ）を充填した。この溶液に、ＴＦＡ（２．５ｍＬ）を室温で添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：Ｃ１８、２５０×２１．２ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％トリフルオロ酢酸、Ｂ＝アセトニトリル、Ａ：Ｂ＝６５：２５、保持時間４分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．０１４ｇ、５．９５％）。ＬＣＭＳ：８９４．４［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３７（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ７．２５（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０ ＆ １．６Ｈｚ １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４６（ｓ， アセタール－Ｈ， １Ｈ）， ５．０７（ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．６３（ｄ， Ｊ＝１９．６Ｈｚ， １Ｈ）， ４．４５－４．４３（ｍ， ２Ｈ）， ４．３４（ｄ， Ｊ＝１９．６Ｈｚ， １Ｈ）， ４．１０－３．９０（ｍ， ５Ｈ）， ２．９０－１．６０（ｍ， ２４Ｈ）， １．５２（ｓ， ３Ｈ）， １．２５－１．０５（ｍ， ２Ｈ）， １．００（ｓ， ３Ｈ）。 (4S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-1-yl)amino)-5-oxo Synthesis of pentanoic acid (INX-A8)

procedure:
In a 25 mL single neck round bottom flask, (4S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R) , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane- tert-Butyl (1-yl)amino)-5-oxopentanoate (INX-A8-3) (0.250 g, 0.262 mmol) and DCM (15 mL) were charged. To this solution was added TFA (2.5 mL) at room temperature and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was purified by preparative HPLC (column: C18, 250 x 21.2 mm, 5 μm, mobile phase: A = 0.05% trifluoroacetic acid in water, B = acetonitrile, A:B = 65:25, retention time 4 min. ) to give the title compound as an off-white solid (0.014 g, 5.95%). LCMS: 894.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ: 7.47 (d, J = 10.0 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H ), 7.25 (d, J=8.0Hz, 2H), 6.27 (dd, J=10.0 & 1.6Hz 1H), 6.04 (s, 1H), 5.46 (s, Acetal-H, 1H), 5.07 (d, J=5.2Hz, C16H, 1H), 4.63 (d, J=19.6Hz, 1H), 4.45-4.43 (m, 2H) ), 4.34 (d, J=19.6Hz, 1H), 4.10-3.90 (m, 5H), 2.90-1.60 (m, 24H), 1.52 (s, 3H) ), 1.25-1.05 (m, 2H), 1.00 (s, 3H).

２－（４－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）シクロヘキシリデン）酢酸エチル（ＩＮＸ－ＳＭ－４６－１）の合成

手順：
１００ｍＬの一つ口丸底フラスコに、ＮａＨ（１．２ｇ、３０．５ｍｍｏｌ）及びＴＨＦ（５０ｍＬ）を充填した。この溶液に、２－（ジエトキシホスホリル）酢酸エチル（６．８３ｇ、３０．５ｍｍｏｌ）を０℃で添加し、室温で３０分間撹拌した。ＴＨＦ（５ｍＬ）中の（４－オキソシクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（５．０ｇ、２３．４７ｍｍｏｌ）の溶液を反応混合物に添加し、室温で１２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＮＨ_４Ｃｌの飽和溶液（１００ｍＬ）でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、白色の固体として表題化合物を得た。（６．５ｇ、９７．７８％）。ＬＣＭＳ：２８４．１ ［Ｍ＋Ｈ］^＋。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[2.5]octan-1-yl)methyl)phenyl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-46)

Synthesis of ethyl 2-(4-((tert-butoxycarbonyl)amino)cyclohexylidene)acetate (INX-SM-46-1)

procedure:
A 100 mL single neck round bottom flask was charged with NaH (1.2 g, 30.5 mmol) and THF (50 mL). Ethyl 2-(diethoxyphosphoryl)acetate (6.83 g, 30.5 mmol) was added to this solution at 0° C. and stirred at room temperature for 30 minutes. A solution of tert-butyl (4-oxocyclohexyl)carbamate (5.0 g, 23.47 mmol) in THF (5 mL) was added to the reaction mixture and stirred at room temperature for 12 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with a saturated solution of NH ₄ Cl (100 mL) and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a white solid. (6.5g, 97.78%). LCMS: 284.1 [M+H] ⁺ .

手順：
１００ｍＬの一つ口丸底フラスコに、６０％のＮａＨ（０．７ｇ、１７．６ｍｍｏｌ）及びＤＭＳＯ（４０ｍＬ）を充填した。この溶液に、ヨウ化トリメチルスルホキソニウム（３．８ｇ、１７．６ｍｍｏｌ）を０℃で添加し、室温で３０分間撹拌した。ＤＭＳＯ（５ｍＬ）中の２－（４－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）シクロヘキシリデン）酢酸エチル（ＩＮＸ－ＳＭ－４６－１）（２．０ｇ、７．０６ｍｍｏｌ）の溶液を、この溶液に添加し、室温で１２時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物をＮＨ_４Ｃｌの飽和溶液（１００ｍＬ）でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル：ヘキサン、２０：８０）によって精製し、オフホワイト色の固体として表題化合物を得た（０．７５ｇ、３５．７３％）。ＬＣＭＳ ２９８．２ ［Ｍ＋Ｈ］^＋。 Synthesis of ethyl 6-((tert-butoxycarbonyl)amino)spiro[2.5]octane-1-carboxylate (INX-SM-46-2)

procedure:
A 100 mL single neck round bottom flask was charged with 60% NaH (0.7 g, 17.6 mmol) and DMSO (40 mL). To this solution was added trimethylsulfoxonium iodide (3.8 g, 17.6 mmol) at 0° C. and stirred at room temperature for 30 minutes. A solution of ethyl 2-(4-((tert-butoxycarbonyl)amino)cyclohexylidene)acetate (INX-SM-46-1) (2.0 g, 7.06 mmol) in DMSO (5 mL) was added to this solution. and stirred at room temperature for 12 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with a saturated solution of NH ₄ Cl (100 mL) and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate:hexane, 20:80) to give the title compound as an off-white solid (0.75 g, 35.73%). LCMS 298.2 [M+H] ⁺ .

手順：
３５ｍＬのガラスバイアルに、窒素下で６－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）スピロ［２．５］オクタン－１－カルボン酸エチル（ＩＮＸ－ＳＭ－４６－２）（０．１ｇ、０．３３ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ＬｉＢＨ_４（３．３ｍＬ、６７．３ｍｍｏｌ）を０℃で添加し、室温で１２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を希釈ＨＣｌ（３０ｍＬ）でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、５０：５０）によって精製し、無色の液体として表題化合物を得た（０．０５ｇ、５８．２２％）。ＬＣＭＳ：２５６．２ ［Ｍ＋Ｈ］^＋。 Synthesis of tert-butyl (1-(hydroxymethyl)spiro[2.5]octan-6-yl)carbamate (INX-SM-46-3)

procedure:
In a 35 mL glass vial under nitrogen was added ethyl 6-((tert-butoxycarbonyl)amino)spiro[2.5]octane-1-carboxylate (INX-SM-46-2) (0.1 g, 0.33 mmol). ) and THF (5 mL). To this solution was added LiBH ₄ (3.3 mL, 67.3 mmol) at 0° C. and stirred at room temperature for 12 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with dilute HCl (30 mL) and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 50:50) to give the title compound as a colorless liquid (0.05 g, 58.22%). LCMS: 256.2 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（１－（ヒドロキシメチル）スピロ［２．５］オクタン－６－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４６－３）（０．２ｇ、０．７８ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ＤＭＰ（０．４９８ｇ、１．１７ｍｍｏｌ）を室温で添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＮａＨＣＯ３溶液でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をブラインで洗浄し、Ｎａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、黄色の液体として表題化合物を得て（０．２ｇ、粗製）、これを更に精製することなくそのようなものとして次のステップに進めた。 Synthesis of tert-butyl (1-formylspiro[2.5]octan-6-yl)carbamate (INX-SM-46-4)

procedure:
In a 25 mL single-necked round-bottom flask, add tert-butyl (1-(hydroxymethyl)spiro[2.5]octan-6-yl)carbamate (INX-SM-46-3) (0.2 g, 0.0 g). 78 mmol) and THF (5 mL). DMP (0.498 g, 1.17 mmol) was added to this solution at room temperature and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with NaHCO3 solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a yellow liquid (0.2 g, crude), which was purified without further purification. As such, we proceeded to the next step.

(E) Synthesis of tert-butyl-(1-((2-tosylhydrazono)methyl)spiro[2.5]octan-6-yl)carbamate (INX-SM-46-5) INX-SM-46-5
procedure:
In a 10 mL glass vial, tert-butyl (1-formylspiro[2.5]octan-6-yl)carbamate (INX-SM-46-4) (0.85 g, 3.35 mmol) and ethanol (20 mL) were added. filled with. To this solution, p-toluenesulfonyl hydrazide (0.74 g, 4.03 mmol) and acetic acid (0.05 g, 0.88 mmol) were added and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the solid was filtered and dried under vacuum to give the title compound as a white solid (1.0 g, 70.70%). LCMS: 422.3 [M+H] ⁺

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｅ）－（１－（（２－トシルヒドラゾノ）メチル）スピロ［２．５］オクタン－６－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４６－５）（０．５ｇ、１．１８ｍｍｏｌ）、Ｋ_２ＣＯ_３（０．２４ｇ、１．７８ｍｍｏｌ）及びジオキサン（１５ｍＬ）を充填し、混合物をＮ_２で３０分間脱気した。この溶液に、（４－ホルミルフェニル）ボロン酸（０．２６ｇ、１．７８ｍｍｏｌ）を添加し、１１０℃で２時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を水（５０ｍＬ）に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル：ヘキサン、１：１）によって精製し、無色の液体として表題化合物を得た（０．２７ｇ、６７．５％）。ＬＣＭＳ：３４４．２１ ［Ｍ＋Ｈ］^＋ Synthesis of tert-butyl (1-(4-formylbenzyl)spiro[2.5]octan-6-yl)carbamate (INX-SM-46-6)

procedure:
In a 50 mL single-neck round bottom flask, add tert-butyl (E)-(1-((2-tosylhydrazono)methyl)spiro[2.5]octan-6-yl)carbamate (INX-SM-46-5). ) (0.5 g, 1.18 mmol), K ₂ CO ₃ (0.24 g, 1.78 mmol) and dioxane (15 mL) and the mixture was degassed with N ₂ for 30 min. To this solution was added (4-formylphenyl)boronic acid (0.26 g, 1.78 mmol) and heated at 110° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water (50 mL) and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate:hexane, 1:1) to give the title compound as a colorless liquid (0.27 g, 67.5%). LCMS: 344.21 [M+H] ⁺

手順：
３５ｍＬのガラスバイアルに、（１－（４－ホルミルベンジル）スピロ［２．５］オクタン－６－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４６－６）（０．５５ｇ、１．６０ｍｍｏｌ）、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．６０２ｇ、１．６０ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＭｇＳＯ_４（０．９６ｇ、８．０１ｍｍｏｌ）及びＨＣｌＯ_４（０．８０ｇ、８．０１ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和重炭酸ナトリウム溶液でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物の一部（０．１５０ｇ）を分取ＨＰＬＣ（カラム：Ｃ１８－（２５０×２１．２ｍｍ）、５ミクロン、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル：メタノール：ＩＰＡ（６５：２５：１０）、Ａ：Ｂ、７５：２５）保持時間１３．６７分）によって精製し、、表題化合物を得た（０．００５ｇ、２．８％）。ＬＣＭＳ：６０２．４ ［Ｍ＋Ｈ］^＋；
^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３９（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．３１（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０ ＆ ２．４Ｈｚ １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４７（ｓ， アセタール－Ｈ， １Ｈ）， ５．０７（ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．７０－４．６０（ｍ， ２Ｈ）， ４．４５（ｂｒ ｓ， １Ｈ）， ４．３５（ｄ， Ｊ＝ １９．２Ｈｚ， １Ｈ）， ３．００－０．８０（ｍ，２２Ｈ）， １．５１（ｓ， ３Ｈ）， １．００（ｓ， ３Ｈ）， ００．５９－０．５６（ｍ， １Ｈ），．２６－０．２５（ｍ， １Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[2.5]octan-1-yl)methyl)phenyl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-46)

procedure:
In a 35 mL glass vial, tert-butyl (1-(4-formylbenzyl)spiro[2.5]octan-6-yl)carbamate (INX-SM-46-6) (0.55 g, 1.60 mmol) , (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9 , 10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0.602 g, 1.60 mmol) and DCM ( 5 mL). To this solution were added MgSO ₄ (0.96 g, 8.01 mmol) and HClO ₄ (0.80 g, 8.01 mmol) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. A portion of the crude material (0.150 g) was subjected to preparative HPLC (column: C18-(250 x 21.2 mm), 5 microns, mobile phase: A = 0.05% TFA in water, B = acetonitrile:methanol:IPA (65:25:10), A:B, 75:25) retention time 13.67 min) to give the title compound (0.005 g, 2.8%). LCMS: 602.4 [M+H] ⁺ ;
^1H NMR (400 MHz, MeOD): δ: 7.47 (d, J=10.0Hz, 1H), 7.39 (d, J=8.0Hz, 2H), 7.31 (d, J= 8.0Hz, 2H), 6.27 (dd, J=10.0 & 2.4Hz 1H), 6.04 (s, 1H), 5.47 (s, acetal-H, 1H), 5.07 (d, J=5.2Hz, C16H, 1H), 4.70-4.60 (m, 2H), 4.45 (br s, 1H), 4.35 (d, J= 19.2Hz, 1H ), 3.00-0.80 (m, 22H), 1.51 (s, 3H), 1.00 (s, 3H), 00.59-0.56 (m, 1H), . 26-0.25 (m, 1H).

（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［２．５］オクタン－６－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ９－１）の合成

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．３２ｇ、０．６８ｍｍｏｌ）、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［２．５］オクタン－１－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－４６）（０．４ｇ、０．６８ｍｍｏｌ）及びＤＭＦ（５ｍＬ）を充填した。この溶液に、ＤＩＰＥＡ（０．１３ｇ、１．０２ｍｍｏｌ）及びＨＡＴＵ（０．３３ｇ、１．０２ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水、５０：５０）によって精製し、淡黄色の固体として表題化合物を得た（０．３ｇ、４２．３１％）。ＬＣＭＳ：１０６６．４１［Ｍ＋Ｈ］^＋。 (4S)-4-(2-(2-bromoacetamide)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[2.5]octan-6-yl)amino)-5-oxo Synthesis of pentanoic acid (INX-A9)

(4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a , 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[2.5] Synthesis of tert-butyl octan-6-yl)amino)-5-oxopentanoate (INX-A9-1)

procedure:
In a 10 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.32 g, 0.68 mmol), (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro [2.5]octan-1-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b , 11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-46) (0.4 g, 0.68 mmol) and DMF (5 mL). DIPEA (0.13 g, 1.02 mmol) and HATU (0.33 g, 1.02 mmol) were added to this solution and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water, 50:50) to give the title compound as a pale yellow solid (0.3 g, 42.31%). LCMS: 1066.41 [M+H] ⁺ .

手順：
１０ｍＬのガラスバイアルに、（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［２．５］オクタン－６－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ９－１）（０．３ｇ、０．２８ｍｍｏｌ）及びＴＨＦ（３ｍＬ）を充填した。この溶液に、ジエチルアミン（０．２０ｇ、２．８１ｍｍｏｌ）を室温で添加し、３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテル及びペンタンで粉砕して、黄色の固体として表題化合物を得た（０．１６ｇ、６７．４０％）ＬＣＭＳ：８４４．５ ［Ｍ＋Ｈ］^＋。 (4S)-4-(2-aminoacetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[2.5]octan-6-yl)amino)-tert-butyl-5-oxopentanoate Synthesis of (INX-A9-2)

procedure:
In a 10 mL glass vial, (4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((1-(4-((6aR,6bS) ,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8 , 8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl) Tert-butyl spiro[2.5]octan-6-yl)amino)-5-oxopentanoate (INX-A9-1) (0.3 g, 0.28 mmol) and THF (3 mL) were charged. Diethylamine (0.20 g, 2.81 mmol) was added to this solution at room temperature and stirred for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether and pentane to give the title compound as a yellow solid (0.16 g, 67.40%) LCMS: 844. 5 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－アミノアセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［２．５］オクタン－６－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ９－２）（０．１６ｇ、０．１８ｍｍｏｌ）及びＤＣＭ（１０ｍＬ）を充填した。この溶液に、水（１ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０４０ｇ、０．３７ｍｍｏｌ）及びブロモアセチルブロミド（０．０３８ｇ、０．１８ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭ中の１０％メタノールで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水、７０：３０）によって精製し、淡黄色の固体として表題化合物を得た（０．１３ｇ、７４．８％）。ＬＣＭＳ：９６４．４［Ｍ＋Ｈ］^＋。 (4S)-4-(2-(2-bromoacetamide)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[2.5]octan-6-yl)amino)-5-oxo Synthesis of tert-butyl pentanoate (INX-A9-3)

procedure:
In a 25 mL single neck round bottom flask, (4S)-4-(2-aminoacetamide)-5-((1-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[2.5]octan-6-yl)amino )-5-oxopentanoate (INX-A9-2) (0.16 g, 0.18 mmol) and DCM (10 mL) were charged. To this solution was added Na ₂ CO ₃ (0.040 g, 0.37 mmol) and bromoacetyl bromide (0.038 g, 0.18 mmol) dissolved in water (1 mL) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with 10% methanol in DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water, 70:30) to give the title compound as a pale yellow solid (0.13 g, 74.8%). LCMS: 964.4 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［２．５］オクタン－６－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ９－３）（０．１３ｇ、０．１３ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．３８ｇ、３．３ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた（０．１１ｇ、粗製）。粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ、６０：４０）、保持時間１４．４分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．００７ｇ、５．７２％）。ＬＣＭＳ：９０８．４０ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４９－７．２７（ｍ， ５Ｈ）， ６．２８（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．０６（ｓ， １Ｈ）， ５．４８（ｓ， アセタール－Ｈ， １Ｈ）， ５．０８（ｄ， Ｊ＝５．２ Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．７０－３．７０（ｍ， ９Ｈ）， ３．００－０．９０（ｍ， ２６Ｈ）， １．５０（ｓ， ３Ｈ）， １．１０（ｓ， ３Ｈ）， ０．５４－０．５１（ｍ， １Ｈ）， ０．１９－０．１７（ｍ， １Ｈ）。 (4S)-4-(2-(2-bromoacetamide)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[2.5]octan-6-yl)amino)-5-oxo Synthesis of pentanoic acid (INX-A9)

procedure:
In a 10 mL single neck round bottom flask, (4S)-4-(2-(2-bromoacetamido)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,10R) , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[2.5]octane- tert-Butyl (6-yl)amino)-5-oxopentanoate (INX-A9-3) (0.13 g, 0.13 mmol) and DCM (2 mL) were charged. To this solution was added TFA (0.38 g, 3.3 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum (0.11 g, crude). The crude material was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B, 60:40), retention time 14. 4 min) to give the title compound as an off-white solid (0.007 g, 5.72%). LCMS: 908.40 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ 7.49-7.27 (m, 5H), 6.28 (d, J=10Hz, 1H), 6.06 (s, 1H), 5.48 (s, acetal-H, 1H), 5.08 (d, J=5.2 Hz, C16H, 1H), 4.70-3.70 (m, 9H), 3.00-0.90 (m, 26H), 1.50 (s, 3H), 1.10 (s, 3H), 0.54-0.51 (m, 1H), 0.19-0. 17 (m, 1H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（４－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［２．２．２］オクタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｚ－１）の合成

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．２０ｇ、０．４１ｍｍｏｌ）を充填し、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（４－アミノビシクロ［２．２．２］オクタン－１－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－１０）（０．２８ｇ、０．４６ｍｍｏｌ）をＤＭＦ（２ｍＬ）に取り込んだ。この溶液に、ＤＩＰＥＡ（０．２１ｇ、１．６ｍｍｏｌ）及びＨＡＴＵ（０．４４ｇ、１．１ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（アセトニトリル／水：５０：５０）によって精製し、淡黄色の固体として表題化合物を得た（０．１ｇ、２０．１６％）。ＬＣＭＳ １０６６．９［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[2.2.2]octan-1-yl)amino)-5 -Synthesis of oxopentanoic acid (INX-Z)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((4-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a , 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[2.2. 2] Synthesis of tert-butyl octan-1-yl)amino)-5-oxopentanoate (INX-Z-1)

procedure:
In a 10 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Filled with oxopentanoic acid (INX-P-4) (0.20 g, 0.41 mmol), (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((4- aminobicyclo[2.2.2]octan-1-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8 ,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX- SM-10) (0.28 g, 0.46 mmol) was taken up in DMF (2 mL). DIPEA (0.21 g, 1.6 mmol) and HATU (0.44 g, 1.1 mmol) were added to this solution and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile/water: 50:50) to give the title compound as a pale yellow solid (0.1 g, 20.16%). LCMS 1066.9 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（４－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［２．２．２］オクタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｚ－１）（０．１５ｇ、０．１４ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ジエチルアミン（０．１０ｇ、１．４ｍｍｏｌ）を添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテル及びペンタンで粉砕して、黄色の固体として表題化合物を得た（０．０８ｇ、６７．３８％）。ＬＣＭＳ：８４４．６ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-aminoacetamide)-5-((4-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[2.2.2]octan-1-yl)amino)-5-oxopentanoic acid tert -Synthesis of butyl (INX-Z-2)

procedure:
In a 10 mL single neck round bottom flask, add (4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((4-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7 , 8,8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl) Filled with tert-butyl (benzyl)bicyclo[2.2.2]octan-1-yl)amino)-5-oxopentanoate (INX-Z-1) (0.15 g, 0.14 mmol) and THF (5 mL). did. To this solution was added diethylamine (0.10 g, 1.4 mmol) and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether and pentane to give the title compound as a yellow solid (0.08 g, 67.38%). LCMS: 844.6 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－アミノアセトアミド）－５－（（４－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［２．２．２］オクタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｚ－２）（０．０８ｇ、０．０９４ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、水（１ｍＬ）中のＮａ_２ＣＯ_３（０．０３０ｇ、０．２８ｍｍｏｌ）溶液、続いて、ブロモアセチルブロミド（０．０２４ｇ、０．１２ｍｍｏｌ）を室温で滴加し、反応混合物を１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、淡黄色の固体として表題化合物を得た（０．０８ｇ、８８．２％）。ＬＣＭＳ：Ｃ５０Ｈ６７^７９ＢｒＮ_３Ｏ_１１に対する計算値（９６４．４０）、実測値９６４．６［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[2.2.2]octan-1-yl)amino)-5 -Synthesis of tert-butyl oxopentanoate (INX-Z-3)

procedure:
In a 10 mL single-neck round bottom flask, (4S)-4-(2-aminoacetamide)-5-((4-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[2.2.2]octan-1-yl)amino )-5-oxopentanoate (INX-Z-2) (0.08 g, 0.094 mmol) and DCM (2 mL) were charged. To this solution was added a solution of Na ₂ CO ₃ (0.030 g, 0.28 mmol) in water (1 mL) followed by bromoacetyl bromide (0.024 g, 0.12 mmol) dropwise at room temperature and the reaction mixture was Stirred for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a pale yellow solid (0.08 g, 88.2%). LCMS: Calcd for C50H67 ⁷⁹ BrN ₃ O ₁₁ (964.40), found 964.6 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（４－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［２．２．２］オクタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｚ－３）（０．０８ｇ、０．０８ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．２３ｇ、２．１０ｍｍｏｌ）を添加し、反応混合物を室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラムＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．１％ＦＡ、Ｂ＝ＡＣＮ：ＭＥＯＨ（５０：５０）、Ａ：Ｂ＝３５：６５、保持時間２０．１７分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．００５ｇ、６．６４％）ＬＣＭＳ：Ｃ_４６Ｈ_５９ ^７９ＢｒＮ_３Ｏ_１１に対する計算値（９０８．３３）、実測値９０８．４［Ｍ＋Ｈ］^＋；
^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ） 主要プロトン割り当て）： ^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４６（ｄ， Ｊ＝１０．４Ｈｚ， １Ｈ）， ７．３４ （ｄ， Ｊ＝７．６Ｈｚ， ２Ｈ）， ７．０９（ｄ， Ｊ＝７．６Ｈｚ， ２Ｈ）， ６．２６（ｄ， Ｊ＝９．６Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４４（ｓ， アセタール－Ｈ， １Ｈ）， ５．０５（ｄ， Ｊ＝５．２ Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ５．００－４．１０（ｍ， ９Ｈ）， ２．７０－１．６０（ｍ， ２０Ｈ）， １．５１ （ｓ， ３Ｈ）， １．５０－１．４０（ｍ， ６Ｈ）， １．２５－１．０５（ｍ， ２Ｈ）， １．００（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[2.2.2]octan-1-yl)amino)-5 -Synthesis of oxopentanoic acid (INX-Z)

procedure:
In a 10 mL single neck round bottom flask, (4S)-4-(2-(2-bromoacetamido)acetamide)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,11aR , 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a , 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[2.2.2]octane- tert-Butyl (1-yl)amino)-5-oxopentanoate (INX-Z-3) (0.08 g, 0.08 mmol) and DCM (2 mL) were charged. To this solution was added TFA (0.23 g, 2.10 mmol) and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude material was subjected to preparative HPLC (column YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.1% FA in water, B = ACN:MEOH (50:50), A:B=35:65, retention _time 20.17 min) to give the title compound as _an off-white solid (0.005 g, ^6.64 %) LCMS: _{C46H5979BrN3O} Calculated value for ₁₁ (908.33), actual value 908.4 [M+H] ⁺ ;
¹ H NMR (400 MHz, MeOD) (major proton assignment): ¹ H NMR (400 MHz, MeOD): δ 7.46 (d, J=10.4 Hz, 1H), 7.34 (d, J=7. 6Hz, 2H), 7.09 (d, J=7.6Hz, 2H), 6.26 (d, J=9.6Hz, 1H), 6.04 (s, 1H), 5.44 (s, Acetal-H, 1H), 5.05 (d, J=5.2 Hz, C16H, 1H), 5.00-4.10 (m, 9H), 2.70-1.60 (m, 20H) , 1.51 (s, 3H), 1.50-1.40 (m, 6H), 1.25-1.05 (m, 2H), 1.00 (s, 3H).

（Ｅ）－（１－（（２－トシルヒドラゾノ）メチル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３６－１）の合成

手順：
５０ｍＬの一つ口丸底フラスコに、窒素下で（１－ホルミル－２－オキサビシクロ［２．２．２］オクタン－４－イル）カルバミン酸ｔｅｒｔ－ブチル（０．２ｇ、０．７８ｍｍｏｌ）及びＥｔＯＨ（５ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホンヒドラジド（０．２１８ｇ、１．１７ｍｍｏｌ）及び触媒量のＡｃＯＨ（０．１ｍＬ）を添加し、反応混合物を室温で０．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、結果として得られた固体を濾過し、真空下で乾燥させて、白色の固体として表題化合物を得た（０．３０ｇ、９０．４２％）。ＬＣＭＳ：４２４．２３［Ｍ＋Ｈ］^＋ (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((4-amino-2-oxabicyclo[2.2.2]octan-1-yl)methyl)phenyl) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[ Synthesis of 2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-36)

(E) Synthesis of tert-butyl-(1-((2-tosylhydrazono)methyl)-2-oxabicyclo[2.2.2]octan-4-yl)carbamate (INX-SM-36-1)

procedure:
In a 50 mL single neck round bottom flask under nitrogen, tert-butyl (1-formyl-2-oxabicyclo[2.2.2]octan-4-yl)carbamate (0.2 g, 0.78 mmol) and Charged with EtOH (5 mL). To this solution, p-toluenesulfone hydrazide (0.218 g, 1.17 mmol) and a catalytic amount of AcOH (0.1 mL) were added and the reaction mixture was stirred at room temperature for 0.5 h. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the resulting solid was filtered and dried under vacuum to give the title compound as a white solid (0.30 g, 90.42 %). LCMS: 424.23 [M+H] ⁺

手順：
３５ｍＬのガラスバイアルに、窒素下で（Ｅ）－（１－（（２－トシルヒドラゾノ）メチル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３６－１）（０．１ｇ、０．２３６ｍｍｏｌ）及びジオキサン（３ｍＬ）を充填した。この溶液に、（４－ホルミルフェニル）ボロン酸（０．０５３ｇ、０．３５４ｍｍｏｌ）及びＫ_２ＣＯ_３（０．０４８ｇ、０．３５４ｍｍｏｌ）を添加し、１００℃で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、３０：７０）によって精製し、黄色の粘着性固体として表題化合物を得た（０．０３０ｇ、３６．７８％）。ＬＣＭＳ：３４６．２ ［Ｍ＋Ｈ］^＋ Synthesis of tert-butyl (1-(4-formylbenzyl)-2-oxabicyclo[2.2.2]octan-4-yl)carbamate (INX-SM-36-2)

procedure:
In a 35 mL glass vial, tert-butyl (E)-(1-((2-tosylhydrazono)methyl)-2-oxabicyclo[2.2.2]octan-4-yl)carbamate (INX- SM-36-1) (0.1 g, 0.236 mmol) and dioxane (3 mL) were charged. To this solution were added (4-formylphenyl)boronic acid (0.053 g, 0.354 mmol) and K ₂ CO ₃ (0.048 g, 0.354 mmol) and stirred at 100° C. for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 30:70) to give the title compound as a yellow sticky solid (0.030 g, 36.78%). LCMS: 346.2 [M+H] ⁺

手順：
５０ｍＬの一つ口丸底フラスコに、（１－（４－ホルミルベンジル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３６－２）（０．０２５ｇ、０．０７２ｍｍｏｌ）、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．０２７ｇ、０．０７２ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＭｇＳＯ_４（０．０４３ｇ、０．３６１５ｍｍｏｌ）及びＨＣｌＯ４（０．０３６ｇ、０．３６１５ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、真空下で濃縮した。粗製物を冷水で粉砕し、参加したものを濾過し、真空下で乾燥させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝７５：２５）、保持時間１２．５２分）によって精製し、白色の固体として表題化合物を得た。（０．００８ｇ、１５．５４％）。ＬＣＭＳ：６０４．４ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３７（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．２２（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０ ＆ １．６Ｈｚ １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４７（ｓ， アセタール－Ｈ， １Ｈ）， ５．０８（ｄ， Ｊ＝４．８Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．６５（ｄ， Ｊ＝ １９．６Ｈｚ， １Ｈ）， ４．４５－４．４０（ｍ １Ｈ）， ４．３３（ｄ， Ｊ＝ １９．６Ｈｚ， １Ｈ）， ３．３０（ｓ， ２Ｈ）， ２．７３（ｓ， ２Ｈ）， ２．７０－２．６９（ｍ， １Ｈ）， ２．５０－１．６０（ｍ， １６Ｈ）， １．５２（ｓ， ３Ｈ）， １．２０－１．０４（ｍ， ２Ｈ）， １．０１（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((4-amino-2-oxabicyclo[2.2.2]octan-1-yl)methyl)phenyl) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[ Synthesis of 2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-36)

procedure:
In a 50 mL single-neck round bottom flask, add tert-butyl (1-(4-formylbenzyl)-2-oxabicyclo[2.2.2]octan-4-yl)carbamate (INX-SM-36-2). ) (0.025g, 0.072mmol), (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13 -dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0 .027 g, 0.072 mmol) and DCM (2 mL). To this solution were added _MgSO4 (0.043g, 0.3615mmol) and HClO4 (0.036g, 0.3615mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated _NaHCO3 solution and concentrated under vacuum. The crude was triturated with cold water and the residue was filtered and dried under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 75: 25), retention time 12.52 min) to give the title compound as a white solid. (0.008g, 15.54%). LCMS: 604.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ: 7.47 (d, J=10.0 Hz, 1H), 7.37 (d, J=8.0 Hz, 2H ), 7.22 (d, J=8.0Hz, 2H), 6.27 (dd, J=10.0 & 1.6Hz 1H), 6.04 (s, 1H), 5.47 (s, Acetal-H, 1H), 5.08 (d, J = 4.8Hz, C16H, 1H), 4.65 (d, J = 19.6Hz, 1H), 4.45-4.40 (m 1H) , 4.33 (d, J= 19.6Hz, 1H), 3.30 (s, 2H), 2.73 (s, 2H), 2.70-2.69 (m, 1H), 2.50 -1.60 (m, 16H), 1.52 (s, 3H), 1.20-1.04 (m, 2H), 1.01 (s, 3H).

（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－キオソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２４－１）の合成

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．２６３ｇ、０．５４６ｍｍｏｌ）、ＨＡＴＵ（０．２０７ｇ、０．５４６ｍｍｏｌ）、及びＤＭＦ（３ｍＬ）を充填した。この溶液に、ＤＩＰＥＡ（０．１４１ｇ、０．１０９ｍｍｏｌ）、続いて、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（４－アミノ－２－オキサビシクロ［２．２．２］オクタン－１－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－３６）（０．３３０ｇ、０．５４６ｍｍｏｌ）を添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（ＭｅＯＨ／ＤＣＭ、１０：９０）によって精製し、白色の固体として表題化合物を得た（０．３ｇ、５１．３８％）。ＬＣＭＳ：１０６８．７［Ｍ＋Ｈ］^＋ ((4S)-4-(2-(2-bromoacetamide)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2.2.2]octan-4-yl)amino )-5-oxopentanoic acid (INX-A24) synthesis

(4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-chioso-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2. 2.2] Synthesis of tert-butyl octan-4-yl)amino)-5-oxopentanoate (INX-A24-1)

procedure:
In a 50 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.263 g, 0.546 mmol), HATU (0.207 g, 0.546 mmol), and DMF (3 mL) were charged. To this solution was added DIPEA (0.141 g, 0.109 mmol) followed by (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((4-amino-2-oxaR, bicyclo[2.2.2]octan-1-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8, 8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM -36) (0.330 g, 0.546 mmol) was added and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was purified by silica gel column chromatography (MeOH/DCM, 10:90) to give the title compound as a white solid (0.3 g, 51.38%). LCMS: 1068.7 [M+H] ⁺

手順：
２５ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－キオソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２４－１）（０．５ｇ、０．４６８ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ジエチルアミン（０．３４２ｇ、４．６８ｍｍｏｌ）を添加し、室温で２．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で濃縮した。粗製物をＤＣＭ及びヘキサンによる粉砕によって精製し、白色の固体として表題化合物を得た（０．３５ｇ、８８．３８％）。ＬＣＭＳ：８４６．５［Ｍ＋Ｈ］^＋。 (4S)-4-(2-aminoacetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1': 4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2.2.2]octan-4-yl)amino)-5-oxopentane Synthesis of tert-butyl acid (INX-A24-2)

procedure:
In a 25 mL single neck round bottom flask, add (4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((1-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-chioso-2,4,6a,6b,7 , 8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl) tert-butyl)-2-oxabicyclo[2.2.2]octan-4-yl)amino)-5-oxopentanoate (INX-A24-1) (0.5 g, 0.468 mmol) and THF ( 5 mL). To this solution was added diethylamine (0.342 g, 4.68 mmol) and stirred at room temperature for 2.5 hours. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated under vacuum. The crude was purified by trituration with DCM and hexane to give the title compound as a white solid (0.35g, 88.38%). LCMS: 846.5 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－アミノアセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２４－２）（０．３６５ｇ、０．４３１ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、水（０．５ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０９１ｇ、０．８６２ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０８７ｇ、０．４３１ｍｍｏｌ）を添加し、室温で０．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（０．２２ｇ、５２．８％）。ＬＣＭＳ：９６６．４ ［Ｍ＋Ｈ］^＋ (4S)-4-(2-(2-bromoacetamide)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2 ',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2.2.2]octan-4-yl)amino) Synthesis of tert-butyl-5-oxopentanoate (INX-A24-3)

procedure:
In a 25 mL single neck round bottom flask, (4S)-4-(2-aminoacetamide)-5-((1-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2.2.2]octane-4 tert-Butyl-yl)amino)-5-oxopentanoate (INX-A24-2) (0.365 g, 0.431 mmol) and DCM (5 mL) were charged. To this solution was added Na ₂ CO ₃ (0.091 g, 0.862 mmol) dissolved in water (0.5 mL) followed by bromoacetyl bromide (0.087 g, 0.431 mmol) at room temperature. Stirred for 5 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as an off-white solid (0.22 g, 52.8%). LCMS:966.4 [M+H] ⁺

手順：
２５ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２４－３）（０．２ｇ、０．２０６ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＴＦＡ（０．５ｍＬ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：Ｘ－ｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ（２５０×１９）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル：ＭｅＯＨ：ＩＰＡ（６５：２５：１０）、Ａ：Ｂ＝７０：３０、保持時間１５．８８分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．０２５ｇ、１３．３２％）。ＬＣＭＳ：Ｃ_４５Ｈ_５７ ^７９ＢｒＮ_３Ｏ_１２に対する計算値（９１０．３１）、実測値９１０．６［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３７（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．２０（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４６（ｓ， アセタール－Ｈ， １Ｈ）， ５．０８（ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．６４（ｄ， Ｊ＝ １９．６Ｈｚ， １Ｈ）， ４．４５－４．４４（ｍ １Ｈ）， ４．３４（ｄ， Ｊ＝ １９．６Ｈｚ， １Ｈ）， ４．２７－４．２２（ｍ， １Ｈ）， ３．９８－３．８９（ｍ， ６Ｈ）， ２．７０（ｓ， ２Ｈ）， ２．５０－１．５５（ｍ， ２１Ｈ）， １．５２（ｓ， ３Ｈ）， １．２０－１．０４（ｍ， ２Ｈ）， １．０１（ｓ， ３Ｈ）。 ((4S)-4-(2-(2-bromoacetamide)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2.2.2]octan-4-yl)amino )-5-oxopentanoic acid (INX-A24) synthesis

procedure:
In a 25 mL single neck round bottom flask, (4S)-4-(2-(2-bromoacetamido)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR , 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a , 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2.2. 2] tert-Butyl octan-4-yl)amino)-5-oxopentanoate (INX-A24-3) (0.2 g, 0.206 mmol) and DCM (5 mL) were charged. To this solution was added TFA (0.5 mL) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude material was subjected to preparative HPLC (column: :10), A:B=70:30, retention time 15.88 min) to give the title compound as an off-white solid (0.025 g, 13.32%). LCMS: Calculated value for C ₄₅ H ₅₇ ⁷⁹ BrN ₃ O ₁₂ (910.31), actual value 910.6 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ: 7.47 (d, J= 10.0Hz, 1H), 7.37 (d, J=8.0Hz, 2H), 7.20 (d, J=8.0Hz, 2H), 6.27 (dd, J=10.0, 1H) ), 6.04 (s, 1H), 5.46 (s, acetal-H, 1H), 5.08 (d, J=5.2Hz, C16H, 1H), 4.64 (d, J= 19 .6Hz, 1H), 4.45-4.44 (m 1H), 4.34 (d, J= 19.6Hz, 1H), 4.27-4.22 (m, 1H), 3.98- 3.89 (m, 6H), 2.70 (s, 2H), 2.50-1.55 (m, 21H), 1.52 (s, 3H), 1.20-1.04 (m, 2H), 1.01(s, 3H).

（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２５－１）の合成

手順：
３５ｍＬのガラスバイアルに、（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－キオソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２４－１）（０．３００ｇ、０．２８０ｍｍｏｌ）及びＤＭＦ（０．６ｍＬ）を充填した。この溶液に、１Ｈ－テトラゾール（０．１９６ｇ、２．８００ｍｍｏｌ）及び（ｔＢｕＯ）_２ＰＮ（ｉＰｒ）_２（１．８６ｇ、６．７４１ｍｍｏｌ）を添加し、室温で２４時間撹拌した。反応混合物を０℃まで冷却し、過酸化水素（３ｍＬ）を反応混合物中に添加した。反応混合物を撹拌し、次いで、真空下で濃縮した。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、７０：３０）によって精製し、白色の固体として表題化合物を得た（０．１４０ｇ、３９．５５％）。ＬＣ／ＭＳ：１２６０．７ ［Ｍ＋Ｈ］^＋ (4S)-4-(2-(2-bromoacetamide)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 6a,8a-dimethyl-4-oxo-8b-(2-(phosphonoxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H- naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2.2.2]octan-4-yl ) Synthesis of amino)-5-oxopentanoic acid (INX-A25)

(4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS , 11aR, 12aS, 12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7 , 8,8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl) Synthesis of tert-butyl)-2-oxabicyclo[2.2.2]octan-4-yl)amino)-5-oxopentanoate (INX-A25-1)

procedure:
In a 35 mL glass vial, (4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((1-(4-((6aR,6bS) ,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-chioso-2,4,6a,6b,7,8,8a ,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2 - tert-butyl oxabicyclo[2.2.2]octan-4-yl)amino)-5-oxopentanoate (INX-A24-1) (0.300 g, 0.280 mmol) and DMF (0.6 mL) filled with. To this solution were added 1H-tetrazole (0.196 g, 2.800 mmol) and (tBuO) ₂ PN(iPr) ₂ (1.86 g, 6.741 mmol) and stirred at room temperature for 24 hours. The reaction mixture was cooled to 0° C. and hydrogen peroxide (3 mL) was added into the reaction mixture. The reaction mixture was stirred and then concentrated under vacuum. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 70:30) to give the title compound as a white solid (0.140g, 39.55%). LC/MS: 1260.7 [M+H] ⁺

手順：
３５ｍＬのガラスバイアルに、（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２５－１）（０．１４０ｇ、０．１１１ｍｍｏｌ）及びＥｔＯＡｃ（２ｍＬ）を充填した。この溶液に、ジエチルアミン（０．０８１ｇ、１．１１１ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＥｔＯＡｃで希釈し、Ｈ_２Ｏで洗浄した。有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で濃縮した。粗製物をヘキサンで粉砕し、白色の固体として表題化合物を得た（０．０８０ｇ、６９．３７％）。ＬＣ／ＭＳ：１０３９．０ ［Ｍ＋Ｈ］^＋ (4S)-4-(2-aminoacetamide)--5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-((di -tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2.2.2]octane- Synthesis of tert-butyl (4-yl)amino)-5-oxopentanoate (INX-A25-2)

procedure:
In a 35 mL glass vial, (4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((1-(4-((6aR,6bS) ,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4 , 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3] tert-butyl dioxol-10-yl)benzyl)-2-oxabicyclo[2.2.2]octan-4-yl)amino)-5-oxopentanoate (INX-A25-1) (0.140 g, 0 .111 mmol) and EtOAc (2 mL). Diethylamine (0.081 g, 1.111 mmol) was added to this solution and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with EtOAc and washed with _H2O . The organic layer was dried over Na ₂ SO ₄ and concentrated under vacuum. The crude was triturated with hexane to give the title compound as a white solid (0.080 g, 69.37%). LC/MS: 1039.0 [M+H] ⁺

手順：
３５ｍＬのガラスバイアルに、（４Ｓ）－４－（２－アミノアセトアミド）－－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２５－２）（０．０８０ｇ、０．０７７ｍｍｏｌ）及びＤＣＭ（０．９ｍＬ）を充填した。この溶液に、水（０．１ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０１７ｇ、０．１５４ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０１６ｇ、０．０７７ｍｍｏｌ）を室温で添加した。反応混合物を室温で３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で濃縮した（０．０８０ｇ、８９．５７％）。ＬＣ／ＭＳ：１１６０．４ ［Ｍ＋Ｈ］^＋ (4S)-4-(2-(2-bromoacetamide)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2 -((di-tert-butoxyphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2.2.2 ] Synthesis of tert-butyl octan-4-yl)amino)-5-oxopentanoate (INX-A25-3)

procedure:
In a 35 mL glass vial, (4S)-4-(2-aminoacetamide)--5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b- (2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2. 2.2] tert-butyl octan-4-yl)amino)-5-oxopentanoate (INX-A25-2) (0.080 g, 0.077 mmol) and DCM (0.9 mL) were charged. To this solution was added Na ₂ CO ₃ (0.017 g, 0.154 mmol) dissolved in water (0.1 mL) followed by bromoacetyl bromide (0.016 g, 0.077 mmol) at room temperature. The reaction mixture was stirred at room temperature for 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and concentrated under vacuum (0.080 g, 89.57%). LC/MS: 1160.4 [M+H] ⁺

手順：
２５ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（１－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－オキサビシクロ［２．２．２］オクタン－４－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２５－３）（０．０８０ｇ、０．０６９ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．２ｍＬ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ、７０：３０）、保持時間１２．８８分）によって精製し、白色の固体として表題化合物を得た（０．００７ｇ、１０．２４％）。ＬＣＭＳ：Ｃ_４５Ｈ_５８ ^７９ＢｒＮ_３Ｏ_１５Ｐに対する計算値（９９０．２８）、実測値９９０．５［Ｍ＋Ｈ］^＋；
^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．５１－７．４７（ｍ， １Ｈ）， ７．３６ （ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ７．２０（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０ ＆ １．６Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．５２（ｓ， アセタール－Ｈ， １Ｈ）， ５．０７（ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．８５－４．８０（ｍ， １Ｈ）。４．５０－４．４０（ｍ， １Ｈ）， ４．３５－４．２０（ｍ， １Ｈ）， ４．００－３．８０（ｍ， ６Ｈ）， ２．７０－１．５５（ｍ， ２４Ｈ）， １．５２（ｓ， ３Ｈ）， １．２５－１．０５（ｍ， ２Ｈ）， １．０３（ｓ， ３Ｈ） (4S)-4-(2-(2-bromoacetamide)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 6a,8a-dimethyl-4-oxo-8b-(2-(phosphonoxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H- naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-oxabicyclo[2.2.2]octan-4-yl ) Synthesis of amino)-5-oxopentanoic acid (INX-A25)

procedure:
In a 25 mL single neck round bottom flask, (4S)-4-(2-(2-bromoacetamido)acetamide)-5-((1-(4-((6aR,6bS,7S,8aS,8bS,11aR , 12aS, 12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8 , 8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl) tert-butyl-2-oxabicyclo[2.2.2]octan-4-yl)amino)-5-oxopentanoate (INX-A25-3) (0.080 g, 0.069 mmol) and DCM (2 mL) filled with. To this solution was added TFA (0.2 mL) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B, 70: 30), retention time 12.88 min) to give the title compound as a white solid (0.007 g, 10.24%). LCMS: Calculated value _{for C45H5879BrN3O15P} ( _990.28 ), actual _value 990.5 [ ^M +H] ⁺ ;
^1H NMR (400 MHz, MeOD): δ: 7.51-7.47 (m, 1H), 7.36 (d, J=8.4Hz, 2H), 7.20 (d, J=8. 0Hz, 2H), 6.27 (dd, J=10.0 & 1.6Hz, 1H), 6.04 (s, 1H), 5.52 (s, acetal-H, 1H), 5.07 ( d, J=5.2Hz, C16H, 1H), 4.85-4.80(m, 1H). 4.50-4.40 (m, 1H), 4.35-4.20 (m, 1H), 4.00-3.80 (m, 6H), 2.70-1.55 (m, 24H) ), 1.52 (s, 3H), 1.25-1.05 (m, 2H), 1.03 (s, 3H)

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（３－フルオロ－４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１７－１）の合成

手順：
３５ｍＬのガラスバイアルに、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）オキシ）－２－フルオロフェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－Ａ５－１）（０．３ｇ、０．２８０ｍｍｏｌ）及びＤＭＦ（１ｍＬ）を充填した。この溶液に、１Ｈ－テトラゾール（０．１９ｇ、２．８０ｍｍｏｌ）及び（ｔＢｕＯ）_２ＰＮ（ｉＰｒ）_２（１．８ｇ、６．７２ｍｍｏｌ）を室温で添加し、１６時間撹拌した。ＴＬＣによって示される反応の完了後、過酸化水素（３ｍｌ）を０℃で添加し、２時間撹拌した。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、８０：２０）にかけて、薄黄色の固体として表題化合物を得た（０．１８ｇ、５０．８４％）。ＬＣＭＳ：１２６４．５［Ｍ＋Ｈ］^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(3-fluoro-4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS )-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino) Synthesis of -5-oxopentanoic acid (INX-A17)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(3-fluoro-4-((6aR,6bS,7S , 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a ,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[ 3.3] Synthesis of tert-butyl heptan-2-yl)amino)-5-oxopentanoate (INX-A17-1)

procedure:
In a 35 mL glass vial, (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)oxy)- 2-fluorophenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -4H naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-A5-1) (0.3 g, 0.280 mmol) and DMF (1 mL). To this solution were added 1H-tetrazole (0.19 g, 2.80 mmol) and (tBuO) ₂ PN(iPr) ₂ (1.8 g, 6.72 mmol) at room temperature and stirred for 16 hours. After completion of the reaction as indicated by TLC, hydrogen peroxide (3 ml) was added at 0° C. and stirred for 2 hours. The crude was subjected to reverse phase column chromatography (acetonitrile:water, 80:20) to give the title compound as a pale yellow solid (0.18g, 50.84%). LCMS: 1264.5 [M+H] ⁺

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（３－フルオロ－４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１７－１）（０．１８ｇ、０．１７ｍｍｏｌ）及びＴＨＦ（１ｍＬ）を充填した。この溶液に。ジエチルアミン（０．１２５ｇ、１．７０ｍｍｏｌ）を添加し、反応混合物を室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物をジエチルエーテル及びヘキサンで粉砕し、黄色の固体として表題化合物を得た（０．１５ｇ、粗製）。ＬＣＭＳ：１０４２．９［Ｍ＋Ｈ］^＋ (S)-4-(2-aminoacetamide)-5-((6-(3-fluoro-4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentane Synthesis of tert-butyl acid (INX-A17-2)

procedure:
In a 35 mL glass vial, (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((6-(3-fluoro-4-( (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- Filled with tert-butyl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A17-1) (0.18 g, 0.17 mmol) and THF (1 mL). did. into this solution. Diethylamine (0.125g, 1.70mmol) was added and the reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude was triturated with diethyl ether and hexane to give the title compound as a yellow solid (0.15 g, crude). LCMS: 1042.9 [M+H] ⁺

手順：
１０ｍＬのガラスバイアルに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（６－（３－フルオロ－４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１７－２）（０．１３ｇ、０．１４ｍｍｏｌ）及びＤＣＭ（１ｍＬ）を充填した。この溶液に、水（０．３ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０３０ｇ、０．２８ｍｍｏｌ）、続いて。ブロモアセチルブロミド（０．０４３ｇ、０．２１ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（０．１２ｇ、７３．６９％）ＬＣＭＳ：１１６２．６［Ｍ＋Ｈ］^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(3-fluoro-4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS )-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino) Synthesis of tert-butyl-5-oxopentanoate (INX-A17-3)

procedure:
In a 10 mL glass vial, (S)-4-(2-aminoacetamide)-5-((6-(3-fluoro-4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino )-5-oxopentanoate (INX-A17-2) (0.13 g, 0.14 mmol) and DCM (1 mL) were charged. This solution was followed by Na ₂ CO ₃ (0.030 g, 0.28 mmol) dissolved in water (0.3 mL). Bromoacetyl bromide (0.043 g, 0.21 mmol) was added and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as an off-white solid (0.12 g, 73.69%) LCMS: 1162.6 [M+H] ⁺

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（３－フルオロ－４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１７－３）（０．１ｇ、０．０８５ｍｍｏｌ）及びＤＣＭ（１ｍＬ）を充填した。この溶液に、ＴＦＡ（０．０４９ｇ、０．４２ｍｍｏｌ）を室温で添加し、２０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ、６７：３３、保持時間１０．３３分）によって精製し、白色の固体として表題化合物を得た（０．０１０ｇ、１１．７１％）。ＬＣＭＳ：９９４．３［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４９－７．４２（ｍ， ２Ｈ）， ６．６８ （ｄｄ， Ｊ＝８．８ ＆ ２Ｈｚ， １Ｈ）， ６．５８－６．５５（ｍ， １Ｈ）， ６．２８－６．２５（ｄｄ， Ｊ＝１０ ＆ １．６ Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．６９（ｓ， １Ｈ）， ５．０３ （ｄ， Ｊ＝５．２Ｈｚ， １Ｈ）， ４．９５－４．７４（ｍ， ２Ｈ）， ４．６３－４．６０（ｍ， １Ｈ）， ４．４５－４．４４（ｍ， １Ｈ）， ４．３５－４．３１（ｍ， １Ｈ）， ４．２４－４．１８（ｍ， １Ｈ）， ３．９５－３．９０（ｍ， ４Ｈ）， ２．６９－１．７２（ｍ， ２１Ｈ）， １．５２（ｓ， ３Ｈ）， １．３１－１．０５（ｍ， ２Ｈ）， １．０２（ｍ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(3-fluoro-4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS )-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino) Synthesis of -5-oxopentanoic acid (INX-A17)

procedure:
In a 10 mL single-neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(3-fluoro-4-((6aR,6bS,7S,8aS) ,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b , 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3. 3] tert-Butyl heptane-2-yl)amino)-5-oxopentanoate (INX-A17-3) (0.1 g, 0.085 mmol) and DCM (1 mL) were charged. To this solution was added TFA (0.049 g, 0.42 mmol) at room temperature and stirred for 20 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B, 67: 33, retention time 10.33 min) to give the title compound as a white solid (0.010 g, 11.71%). LCMS: 994.3 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ: 7.49-7.42 (m, 2H), 6.68 (dd, J=8.8 & 2Hz, 1H ), 6.58-6.55 (m, 1H), 6.28-6.25 (dd, J=10 & 1.6 Hz, 1H), 6.04 (s, 1H), 5.69 ( s, 1H), 5.03 (d, J=5.2Hz, 1H), 4.95-4.74 (m, 2H), 4.63-4.60 (m, 1H), 4.45- 4.44 (m, 1H), 4.35-4.31 (m, 1H), 4.24-4.18 (m, 1H), 3.95-3.90 (m, 4H), 2. 69-1.72 (m, 21H), 1.52 (s, 3H), 1.31-1.05 (m, 2H), 1.02 (m, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）－３－フルオロフェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１８－１）の合成

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）－３－フルオロフェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ５－１）（０．３ｇ、０．２７０ｍｍｏｌ）及びＤＭＦ（１ｍＬ）を充填した。この溶液に、１Ｈ－テトラゾール（０．１８９ｇ、２．７０ｍｍｏｌ）及び（ｔＢｕＯ）_２ＰＮＥｔ_２（１．７ｇ、６．４８ｍｍｏｌ）を添加し、１６時間撹拌した。反応混合物を０℃まで冷却し、過酸化水素（１ｍＬ）を添加した。反応混合物を逆相カラムクロマトグラフィー（アセトニトリル：水、８０：２０）にかけて、淡黄色の固体として表題化合物を得た（０．１５ｇ、４２．６５％）。ＬＣＭＳ：１３００．５１［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-3-fluorophenoxy)spiro[3. 3] Synthesis of heptane-2-yl)amino)-5-oxopentanoic acid (INX-A18)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS,6bR,7S,8aS ,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ Synthesis of tert-butyl 1,3]dioxol-10-yl)-3-fluorophenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A18-1)

procedure:
In a 35 mL glass vial, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS) , 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4 , 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3] tert-butyl dioxol-10-yl)-3-fluorophenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A5-1) (0.3 g, 0.270 mmol ) and DMF (1 mL). To this solution were added 1H-tetrazole (0.189 g, 2.70 mmol) and (tBuO) ₂ PNEt ₂ (1.7 g, 6.48 mmol) and stirred for 16 hours. The reaction mixture was cooled to 0° C. and hydrogen peroxide (1 mL) was added. The reaction mixture was subjected to reverse phase column chromatography (acetonitrile:water, 80:20) to give the title compound as a pale yellow solid (0.15g, 42.65%). LCMS: 1300.51 [M+H] ⁺ .

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）－３－フルオロフェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１８－１）（０．１２ｇ、０．０９ｍｍｏｌ）及びＴＨＦ（１ｍＬ）を充填した。この溶液に、ジエチルアミン（０．０６７ｇ、０．９２ｍｍｏｌ）を添加し、室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を濃縮し、固体をジエチルエーテル及びヘキサンで粉砕して、黄色の固体として表題化合物を得た（０．０９ｇ、９２．７４％）。ＬＣＭＳ：１０７８．８［Ｍ＋Ｈ］^＋。 (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS,6bR,7S,8aS ,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ Synthesis of tert-butyl 1,3]dioxol-10-yl)-3-fluorophenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A18-2)

procedure:
In a 35 mL glass vial, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR) , 12aS, 12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a ,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole- tert-butyl (10-yl)-3-fluorophenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A18-1) (0.12 g, 0.09 mmol) and Charged with THF (1 mL). To this solution was added diethylamine (0.067 g, 0.92 mmol) and stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated and the solid was triturated with diethyl ether and hexane to give the title compound as a yellow solid (0.09 g, 92.74%). LCMS: 1078.8 [M+H] ⁺ .

手順：
１０ｍＬのガラスバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）－３－フルオロフェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１８－２）（０．０９０ｇ、０．０８３ｍｍｏｌ）及びＤＣＭ（１ｍＬ）を充填した。この溶液に、水（０．３ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０１７ｇ、０．１６６ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０２５ｇ、０．１２ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として表題化合物を含む粗生成物を得た（０．０９０ｇ、９０．４２％）ＬＣＭＳ：１１９９．６［Ｍ＋Ｈ］^＋。 (S)-4-(2-aminoacetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b-(2- ((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-3-fluorophenoxy)spiro [3.3] Synthesis of tert-butyl heptan-2-yl)amino)-5-oxopentanoate (INX-A18-3)

procedure:
In a 10 mL glass vial, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS) ,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a -dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1 ,2-d][1,3]dioxol-10-yl)-3-fluorophenoxy)spiro[3.3]heptan-2-yl)amino)-tert-butyl-5-oxopentanoate (INX-A18- 2) (0.090 g, 0.083 mmol) and DCM (1 mL) were charged. To this solution was added Na ₂ CO ₃ (0.017 g, 0.166 mmol) dissolved in water (0.3 mL) followed by bromoacetyl bromide (0.025 g, 0.12 mmol) for 1 h at room temperature. Stirred. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product containing the title compound as an off-white solid (0.090 g, 90.42%) LCMS: 1199 .6 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）－３－フルオロフェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１８－３）（０．０９０ｇ、０．０７５ｍｍｏｌ）及びＤＣＭ（１ｍＬ）を充填した。この溶液に、ＴＦＡ（０．０４２ｇ、０．３７ｍｍｏｌ）を添加し、室温で２０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ、６７：３３、保持時間１１．３２分）によって精製し、白色の固体として表題化合物を得た（０．０１０ｇ、１２．９３％）。ＬＣＭＳ：１０３１．２［Ｍ＋Ｈ］^＋。^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４４（ｔ， Ｊ＝８．４Ｈｚ， １Ｈ）， ７．３６（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．６８（ｍ， Ｊ＝８．４ ＆ ２ Ｈｚ，１Ｈ）， ６．６０－６．５５（ｍ， １Ｈ）， ６．３７－６．３３（ｍ， ２Ｈ）， ５．７３（ｓ， アセタール－Ｈ， １Ｈ）， ５．６６－５．４９（ｍ， ＣＨ－Ｆ， １Ｈ）， ５．０５ （ｄ， Ｊ＝４．４Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．８０－４．７４（ｍ， ２Ｈ）， ４．６３－４．６０（ｍ， １Ｈ）， ４．３５－４．３１（ｍ， ２Ｈ）， ４．３０－４．２６（ｍ， １Ｈ）， ３．９５－３．９０（ｍ， ４Ｈ）， ２．６８－１．５５（ｍ， ２０Ｈ）， １．５０（ｓ， ３Ｈ）， １．０２（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-3-fluorophenoxy)spiro[3. 3] Synthesis of heptane-2-yl)amino)-5-oxopentanoic acid (INX-A18)

procedure:
In a 10 mL single neck round bottom flask, (S)-4-(2-aminoacetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a, 6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10 tert-butyl)-3-fluorophenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A18-3) (0.090 g, 0.075 mmol) and DCM (1 mL). To this solution was added TFA (0.042 g, 0.37 mmol) and stirred at room temperature for 20 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B, 67: 33, retention time 11.32 minutes) to give the title compound as a white solid (0.010 g, 12.93%). LCMS: 1031.2 [M+H] ⁺ . ^1H NMR (400 MHz, MeOD): δ 7.44 (t, J=8.4Hz, 1H), 7.36 (d, J=10Hz, 1H), 6.68 (m, J=8.4 & 2 Hz, 1H), 6.60-6.55 (m, 1H), 6.37-6.33 (m, 2H), 5.73 (s, acetal-H, 1H), 5.66- 5.49 (m, CH-F, 1H), 5.05 (d, J=4.4Hz, C16H, 1H), 4.80-4.74 (m, 2H), 4.63-4.60 (m, 1H), 4.35-4.31 (m, 2H), 4.30-4.26 (m, 1H), 3.95-3.90 (m, 4H), 2.68-1 .55 (m, 20H), 1.50 (s, 3H), 1.02 (s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ７－１）の合成

手順：
３５ｍＬのガラスバイアルに、Ｎ_２下で（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（１．１４ｇ、２．３８５ｍｍｏｌ）、ＨＡＴＵ（１．３５ｇ、３．５７７ｍｍｏｌ）、及びＤＭＦ（１４ｍＬ）を充填した。この溶液に、ＤＩＰＥＡ（０．８ｍｌ、４．７７ｍｍｏｌ）、続いて、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－３２）（１．４ｇ、２．３８５ｍｍｏｌ）を室温で添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、固体を濾過し、真空下で乾燥させた。粗製物をシリカゲルカラムクロマトグラフィーによって精製し（ＭｅＯＨ：ＤＣＭ ５：９５）、白色の固体として表題化合物を得た（１．６ｇ、６３．８４％）。ＬＣＭＳ：１０５２．６［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H -naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5 -Synthesis of oxopentanoic acid (INX-A7)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a ,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro)[3.3 ] Synthesis of tert-butyl heptane-2-yl)amino)-5-oxopentanoate (INX-A7-1)

procedure:
In a 35 mL glass vial, add (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5- under _N2 . Oxopentanoic acid (INX-P-4) (1.14 g, 2.385 mmol), HATU (1.35 g, 3.577 mmol), and DMF (14 mL) were charged. To this solution was added DIPEA (0.8 ml, 4.77 mmol) followed by (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3 .3] heptan-2-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a , 12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-32) (1 .4 g, 2.385 mmol) was added at room temperature and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the solid was filtered and dried under vacuum. The crude was purified by silica gel column chromatography (MeOH:DCM 5:95) to give the title compound as a white solid (1.6 g, 63.84%). LCMS: 1052.6 [M+H] ⁺ .

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチルＩＮＸ－Ａ７－１）（１．６ｇ、１．５２０ｍｍｏｌ）及びＤＭＦ（２．５ｍＬ）を充填した。この溶液に、１Ｈ－テトラゾール（１．０６ｇ、１５．２０ｍｍｏｌ）及び（ｔＢｕＯ）_２ＰＮ（ｉ－Ｐｒ）_２（１０．１１ｇ、３６．５０１ｍｍｏｌ）を室温で添加し、２４時間撹拌した。反応混合物を℃まで冷却し、過酸化水素（１６ｍＬ）を反応混合物中に添加し、室温で１時間撹拌した。反応混合物を逆相カラムクロマトグラフィー（アセトニトリル：水、１５：８５）にかけて、白色の固体として表題化合物を得た（１．２ｇ、６３．４２％）。ＬＣＭＳ：１２４４．６［Ｍ＋Ｈ］^＋。 (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS ,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- Synthesis of tert-butyl)benzyl)spiro)[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A7-2)

procedure:
In a 35 mL glass vial, (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((6aR,6bS) ,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8 , 8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl) Tert-butyl spiro)[3.3]heptan-2-yl)amino)-5-oxopentanoate INX-A7-1) (1.6 g, 1.520 mmol) and DMF (2.5 mL) were charged. To this solution were added 1H-tetrazole (1.06 g, 15.20 mmol) and (tBuO) ₂ PN(i-Pr) ₂ (10.11 g, 36.501 mmol) at room temperature and stirred for 24 hours. The reaction mixture was cooled to °C, hydrogen peroxide (16 mL) was added into the reaction mixture and stirred at room temperature for 1 hour. The reaction mixture was subjected to reverse phase column chromatography (acetonitrile:water, 15:85) to give the title compound as a white solid (1.2g, 63.42%). LCMS: 1244.6 [M+H] ⁺ .

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ７－２）（１．２ｇ、０．９６４ｍｍｏｌ）及び酢酸エチル（１２ｍＬ）を充填した。この溶液に、ジエチルアミン（０．７０４ｇ、９．６４ｍｍｏｌ）を添加し、室温で４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、水で洗浄し、有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で濃縮した。粗製物をヘキサンで粉砕し、白色の固体として表題化合物を得た（０．６５０ｇ、６５．９４％）。ＬＣＭＳ：１０２３．９［Ｍ＋Ｈ］^＋。 (S)-4-(2-aminoacetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-(( di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro)[3.3]heptan-2-yl) Synthesis of tert-butyl amino)-5-oxopentanoate (INX-A7-3)

procedure:
In a 50 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4 -oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d ][1,3]dioxol-10-yl)benzyl)spiro)[3.3]heptan-2-yl)amino)-tert-butyl-5-oxopentanoate (INX-A7-2) (1.2g, 0.964 mmol) and ethyl acetate (12 mL). To this solution was added diethylamine (0.704 g, 9.64 mmol) and stirred at room temperature for 4 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate, washed with water, and the organic layer was dried over Na ₂ SO ₄ and concentrated under vacuum. The crude was triturated with hexane to give the title compound as a white solid (0.650 g, 65.94%). LCMS: 1023.9 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ７－３）（０．６５０ｇ、０．６３６ｍｍｏｌ）及びＤＣＭ（９ｍＬ）を充填した。この溶液に、水（１．０ｍＬ）に溶解したＮａ_２ＣＯ_３（０．１３５ｇ、１．２７２ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．１２８ｇ、０．６３６ｍｍｏｌ）を室温で添加し、３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、濃縮した。粗製物を逆相カラムクロマトグラフィーによって精製し（アセトニトリル：水、４０：６０）、黄色の固体として表題化合物を得た（０．２５ｇ、３４．７９％）。ＬＣＭＳ：１１４２．６［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b- (2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro)[3.3]heptane Synthesis of tert-butyl-2-yl)amino)-5-oxopentanoate (INX-A7-4)

procedure:
In a 25 mL single neck round bottom flask, (S)-4-(2-aminoacetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a, 8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro)[ 3.3] tert-butyl heptane-2-yl)amino)-5-oxopentanoate (INX-A7-3) (0.650 g, 0.636 mmol) and DCM (9 mL) were charged. To this solution was added Na ₂ CO ₃ (0.135 g, 1.272 mmol) dissolved in water (1.0 mL) followed by bromoacetyl bromide (0.128 g, 0.636 mmol) at room temperature for 30 min. Stirred. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and concentrated. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 40:60) to give the title compound as a yellow solid (0.25g, 34.79%). LCMS: 1142.6 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ７－４）（０．２５０ｇ、０．２１８ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＴＦＡ（０．５ｍＬ）を添加し、反応混合物を室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝５５：４５、保持時間１９．１５分）によって精製し、白色の固体として表題化合物を得た（０．０２６ｇ、１２．２０％）。ＬＣＭＳ：９７４．３［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３６（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．１６（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０ ＆１．６Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．５１（ｓ， アセタール－Ｈ， １Ｈ）， ５．０６ （ｄ， Ｊ＝ ４．８Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ５．００－－４．７０（ｍ， ２Ｈ）， ４．４６－４．４５（ｍ， １Ｈ）， ４．３３－４．３０（ｍ， １Ｈ）， ４．１５－４．０９（ｍ， １Ｈ）， ３．９４－３．９２（ｍ， ３Ｈ）， ２．６８－１．７２（ｍ， ２０Ｈ）， １．５２（ｓ， ３Ｈ）， １．１８－１．０５（ｍ， ２Ｈ）， １．０３（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H -naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5 -Synthesis of tert-butyl oxopentanoate (INX-A7)

procedure:
In a 25 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R) , 11aR, 12aS, 12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7 , 8,8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl) tert-Butyl (benzyl)spiro)[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A7-4) (0.250 g, 0.218 mmol) and DCM (5 mL) were charged. . To this solution was added TFA (0.5 mL) and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 55: 45, retention time 19.15 min) to give the title compound as a white solid (0.026 g, 12.20%). LCMS: 974.3 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ: 7.47 (d, J = 10.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H ), 7.16 (d, J=8.0Hz, 2H), 6.27 (dd, J=10.0 &1.6Hz, 1H), 6.04 (s, 1H), 5.51 (s, Acetal-H, 1H), 5.06 (d, J= 4.8Hz, C16H, 1H), 5.00--4.70 (m, 2H), 4.46-4.45 (m, 1H) , 4.33-4.30 (m, 1H), 4.15-4.09 (m, 1H), 3.94-3.92 (m, 3H), 2.68-1.72 (m, 20H), 1.52 (s, 3H), 1.18-1.05 (m, 2H), 1.03 (s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１２－１）の合成

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２３－１）（１．２ｇ、１．１０ｍｍｏｌ）及びＤＭＦ（２．０ｍＬ）を充填した。この溶液に、１Ｈ－テトラゾール（０．７７１ｇ、１１．０２０ｍｍｏｌ）及び（ｔＢｕＯ）_２ＰＮ（ｉ－Ｐｒ）_２（７．３３ｇ、２６．４６ｍｍｏｌ）を添加し、室温で２４時間撹拌した。反応混合物を０℃まで冷却し、過酸化水素（１２ｍＬ）を反応混合物中に添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を逆相カラムクロマトグラフィー（アセトニトリル：水、７０：３０）にかけて、白色の固体として表題化合物を得た（１．１ｇ、７７．９１％）。ＬＣ／ＭＳ：１２８０．８ ［Ｍ＋Ｈ］^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro)[3.3]heptane Synthesis of -2-yl)amino)-5-oxopentanoic acid (INX-A12)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS,6bR,7S,8aS ,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ Synthesis of tert-butyl 1,3]dioxol-10-yl)benzyl)spiro)[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A12-1)

procedure:
In a 35 mL glass vial, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS) , 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4 , 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3] tert-butyl dioxol-10-yl)benzyl)spiro)[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A23-1) (1.2g, 1.10mmol) and DMF (2.0 mL). To this solution were added 1H-tetrazole (0.771 g, 11.020 mmol) and (tBuO) ₂ PN(i-Pr) ₂ (7.33 g, 26.46 mmol) and stirred at room temperature for 24 hours. The reaction mixture was cooled to 0° C. and hydrogen peroxide (12 mL) was added into the reaction mixture and stirred for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was subjected to reverse phase column chromatography (acetonitrile:water, 70:30) to give the title compound as a white solid (1.1 g, 77.91%). LC/MS: 1280.8 [M+H] ⁺

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１２－１）（１．１ｇ、０．８５９ｍｍｏｌ）及びＥｔＯＡｃ（１１ｍＬ）を充填した。この溶液に、ジエチルアミン（０．６２８ｇ、８．５９３ｍｍｏｌ）を室温で添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＥｔＯＡｃで希釈し、水で洗浄した。有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で濃縮した。粗製物をヘキサンで粉砕し、真空下で乾燥させて、白色の固体として表題化合物を得た（０．５５０ｇ、６０．５０％）。ＬＣ／ＭＳ：１０５８．８ ［Ｍ＋Ｈ］^＋ (S)-4-(2-aminoacetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b-(2- ((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro)[3. 3] Synthesis of tert-butyl heptane-2-yl)amino)-5-oxopentanoate (INX-A12-2)

procedure:
In a 35 mL glass vial, (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS) ,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a -dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1 ,2-d][1,3]dioxol-10-yl)benzyl)spiro)[3.3]heptan-2-yl)amino)-tert-butyl-5-oxopentanoate (INX-A12-1)( 1.1 g, 0.859 mmol) and EtOAc (11 mL). To this solution was added diethylamine (0.628 g, 8.593 mmol) at room temperature and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with EtOAc and washed with water. The organic layer was dried over Na ₂ SO ₄ and concentrated under vacuum. The crude was triturated with hexane and dried under vacuum to give the title compound as a white solid (0.550 g, 60.50%). LC/MS: 1058.8 [M+H] ⁺

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１２－２）（０．５５０ｇ、０．５１９ｍｍｏｌ）及びＤＣＭ（９ｍＬ）を充填した。この溶液に、水（１．０ｍＬ）に溶解したＮａ_２ＣＯ_３（０．１１０ｇ、１．０３８ｍｍｏｌ）及びブロモアセチルブロミド（０．１０５ｇ、０．０．５１９ｍｍｏｌ）を添加し、反応混合物を室温で３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で濃縮して、表題化合物を得た（０．４００ｇ、６５．２７％）。ＬＣ／ＭＳ：１１８０．２ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8 , 8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl) Synthesis of tert-butyl spiro)[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A12-3)

procedure:
In a 35 mL glass vial, (S)-4-(2-aminoacetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7, 8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl ) tert-butyl spiro)[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A12-2) (0.550 g, 0.519 mmol) and DCM (9 mL) were charged. To this solution were added Na ₂ CO ₃ (0.110 g, 1.038 mmol) and bromoacetyl bromide (0.105 g, 0.0.519 mmol) dissolved in water (1.0 mL) and the reaction mixture was heated at room temperature. Stir for 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and concentrated under vacuum to give the title compound (0.400 g, 65.27%). LC/MS: 1180.2 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１２－３）（０．２５０ｇ、０．２１２ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＴＦＡ（０．５ｍＬ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ、６０：４０）によって精製し、白色の固体として表題化合物を得た（０．０２２ｇ、１０．２７％）。ＬＣＭＳ：１０１０．４［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．３７－７．３４（ｍ， ３Ｈ）， ７．１７ （ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．３８－６．３５（ｍ， ２Ｈ）， ５．７０－５．５５（ｍ， ＣＨＦ， １Ｈ）， ５．５４（ｓ， アセタール－Ｈ， １Ｈ）， ５．０７（ｄ， Ｊ＝４．８Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ５．０５－４．８０（ｍ， ３Ｈ）， ４．３５－４．３０（ｍ， ２Ｈ）， ４．１３－４．０９（ｍ， １Ｈ）， ３．９４－３．８９（ｍ， ３Ｈ）， ２．６８－１．６５（ｍ， ２０Ｈ）， １．６０（ｓ， ３Ｈ）， １．０３（ｓ， ３Ｈ） (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro)[3.3]heptane Synthesis of -2-yl)amino)-5-oxopentanoic acid (INX-A12)

procedure:
In a 25 mL single neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS) ,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2 ,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1, 3] Tert-butyl dioxol-10-yl)benzyl)spiro)[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A12-3) (0.250g, 0.212mmol) and DCM (5 mL). To this solution was added TFA (0.5 mL) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and the crude was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.05% TFA in water , B=acetonitrile, A:B, 60:40) to give the title compound as a white solid (0.022 g, 10.27%). LCMS: 1010.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ 7.37-7.34 (m, 3H), 7.17 (d, J=8.0Hz, 2H), 6 .38-6.35 (m, 2H), 5.70-5.55 (m, CHF, 1H), 5.54 (s, acetal-H, 1H), 5.07 (d, J=4. 8Hz, C16H, 1H), 5.05-4.80 (m, 3H), 4.35-4.30 (m, 2H), 4.13-4.09 (m, 1H), 3.94- 3.89 (m, 3H), 2.68-1.65 (m, 20H), 1.60 (s, 3H), 1.03 (s, 3H)

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１４－１）の合成

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ｓ－３）（０．５ｇ、０．４７１ｍｍｏｌ）及びＤＭＦ（１ｍＬ）を充填した。この溶液に、１Ｈ－テトラゾール（０．３３ｇ、４．７１ｍｍｏｌ）及び（ｔＢｕＯ）_２ＰＮ（ｉ－Ｐｒ）_２（３．１３ｇ、１１．３１ｍｍｏｌ）を室温で添加し、２４時間撹拌した。反応混合物を０℃で冷却し、過酸化水素（１０Ｖ）を添加し、室温で１時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物を逆相カラムクロマトグラフィー（アセトニトリル：水、７０：３０）にかけて、薄黄色の固体として表題化合物を得た（０．２８ｇ、４７．４５％）。ＬＣＭＳ：１２５２．７３ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1] Synthesis of pentan-1-yl)amino)-5-oxopentanoic acid (INX-A14)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((2S,6aS,6bR,7S,8aS ,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ Synthesis of tert-butyl 1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-A14-1)

procedure:
In a 35 mL glass vial, (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((2S,6aS) , 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4 , 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3] tert-butyl dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-S-3) (0.5 g, 0.471 mmol) and DMF (1 mL) was charged. 1H-tetrazole (0.33 g, 4.71 mmol) and (tBuO) ₂ PN(i-Pr) ₂ (3.13 g, 11.31 mmol) were added to this solution at room temperature and stirred for 24 hours. The reaction mixture was cooled to 0°C, hydrogen peroxide (10V) was added and allowed to stir at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was subjected to reverse phase column chromatography (acetonitrile:water, 70:30) to give the title compound as a pale yellow solid (0.28 g, 47.45%). LCMS: 1252.73 [M+H] ⁺ .

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１４－１）（０．２８ｇ、０．２３ｍｍｏｌ）及びＴＨＦ（２．８ｍＬ）を充填した。この溶液に、ジエチルアミン（０．１６８ｇ、２．３１ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で濃縮し、粗製物をジエチルエーテル及びヘキサンによる粉砕によって精製し、黄色の固体として表題化合物を得た（０．１９ｇ、８０．１９％）。ＬＣＭＳ：１０３０．６８ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-aminoacetamide)-5-((3-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b-(2- ((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1 .1] Synthesis of tert-butyl pentan-1-yl)amino)-5-oxopentanoate (INX-A14-2)

procedure:
In a 35 mL glass vial, (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((2S,6aS) ,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a -dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1 ,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-tert-butyl-5-oxopentanoate (INX-A14-1) (0.28 g, 0.23 mmol) and THF (2.8 mL). Diethylamine (0.168 g, 2.31 mmol) was added to this solution and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated under vacuum and the crude was purified by trituration with diethyl ether and hexane to give the title compound as a yellow solid (0.19 g, 80.19%) . LCMS: 1030.68 [M+H] ⁺ .

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（３－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１４－２）（０．１９０ｇ、０．１８ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、水（０．４ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０３８ｇ、０．３６ｍｍｏｌ）及びブロモアセチルブロミド（０．０３６ｇ、０．１８ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗粘着性固体として表題化合物を得た（０．１５ｇ、７２．３９％）ＬＣＭＳ：１１５０．４［Ｍ＋Ｈ］^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8 , 8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl) Synthesis of tert-butyl bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-A14-3)

procedure:
In a 35 mL glass vial, (S)-4-(2-aminoacetamide)-5-((3-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7, 8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl ) tert-butyl bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-A14-2) (0.190 g, 0.18 mmol) and DCM (2 mL) were charged. . To this solution was added Na ₂ CO ₃ (0.038 g, 0.36 mmol) and bromoacetyl bromide (0.036 g, 0.18 mmol) dissolved in water (0.4 mL) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a crude sticky solid (0.15 g, 72.39%) LCMS: 1150.4 [M+H] ⁺

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１４－３）（０．１５ｇ、０．１３０ｍｍｏｌ）及びＤＣＭ（４ｍＬ）を充填した。この溶液に、ＴＦＡ（０．０７４ｇ、０．６５ｍｍｏｌ）を添加し、室温で２０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ、６５：３５）、保持時間１２．７６分）によって精製し、白色の固体として表題化合物を得た（０．０１２ｇ、９．３９％）。ＬＣＭＳ：９８２．３［Ｍ＋Ｈ］^＋， ^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ： δ ８．５５（ｔ， １Ｈ）， ８．３５（ｓ， １Ｈ）， ８．０４（ｄ， １Ｈ）， ７．３５ （ｄ， ２Ｈ），７．２５（ｄ， １Ｈ）， ７．１０（ｄ， ２Ｈ）， ６．３０（ｄ， １Ｈ）， ６．１３（ｓ， １Ｈ）， ５．８０－５．６０（ｍ， １Ｈ）， ５．５０（ｓ， アセタール－Ｈ， １Ｈ）， ４．９６（ｄ， Ｊ＝４．８Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．９０－４．７０（ｍ， ２Ｈ）， ４．３０－４．１０（ｍ， ３Ｈ）， ３．９０（ｓ， ２Ｈ）， ３．７５（ｄ， ２Ｈ）， ２．８０（ｄ， ２Ｈ）， ２．５０－１．５５（ｍ， １８Ｈ）， １．５０（ｓ， ３Ｈ）， １．３０－１．１０（ｍ， ２Ｈ）， ０．９２（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1] Synthesis of pentan-1-yl)amino)-5-oxopentanoic acid (INX-A14)

procedure:
In a 10 mL single-neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS) ,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2 ,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1, 3] Tert-butyl dioxol-10-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-A14-3) (0.15 g, 0.130 mmol ) and DCM (4 mL). To this solution was added TFA (0.074 g, 0.65 mmol) and stirred at room temperature for 20 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude material was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B, 65:35), retention time 12. 76 min) to give the title compound as a white solid (0.012 g, 9.39%). LCMS: 982.3 [M+H] ⁺ , ¹ H NMR (400 MHz, DMSO: δ 8.55 (t, 1H), 8.35 (s, 1H), 8.04 (d, 1H), 7.35 (d, 2H), 7.25 (d, 1H), 7.10 (d, 2H), 6.30 (d, 1H), 6.13 (s, 1H), 5.80-5.60 ( m, 1H), 5.50 (s, acetal-H, 1H), 4.96 (d, J=4.8Hz, C16H, 1H), 4.90-4.70 (m, 2H), 4. 30-4.10 (m, 3H), 3.90 (s, 2H), 3.75 (d, 2H), 2.80 (d, 2H), 2.50-1.55 (m, 18H) , 1.50 (s, 3H), 1.30-1.10 (m, 2H), 0.92 (s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１５－１）の合成

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ４－１）（０．５ｇ、０．４２ｍｍｏｌ）及びＤＭＦ（２ｍＬ）を充填した。この溶液に、１Ｈ－テトラゾール（０．３３ｇ、４．７５ｍｍｏｌ）及び（ｔＢｕＯ）_２ＰＮ（ｉ－Ｐｒ）_２（３．１ｇ、１１．２０ｍｍｏｌ）を室温で添加し、１６時間撹拌した。反応混合物を０℃まで冷却し、過酸化水素（１０Ｖ）を反応混合物に添加し、１時間撹拌した。反応混合物を蒸発させ、逆相カラムクロマトグラフィー（アセトニトリル：水、８０：２０）によって精製して、オフホワイト色の固体として表題化合物を得た（０．３ｇ、５７．３０％）ＬＣＭＳ：１２４７．５０ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H -naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5 -Synthesis of oxopentanoic acid (INX-A15)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS ,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- Synthesis of tert-butyl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A15-1)

procedure:
In a 35 mL glass vial, (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((6aR,6bS) ,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a ,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[ 3.3] tert-butyl heptane-2-yl)amino)-5-oxopentanoate (INX-A4-1) (0.5 g, 0.42 mmol) and DMF (2 mL) were charged. To this solution were added 1H-tetrazole (0.33 g, 4.75 mmol) and (tBuO) ₂ PN(i-Pr) ₂ (3.1 g, 11.20 mmol) at room temperature and stirred for 16 hours. The reaction mixture was cooled to 0° C. and hydrogen peroxide (10V) was added to the reaction mixture and stirred for 1 hour. The reaction mixture was evaporated and purified by reverse phase column chromatography (acetonitrile:water, 80:20) to give the title compound as an off-white solid (0.3g, 57.30%) LCMS: 1247. 50 [M+H] ⁺ .

手順：
２５ｍＬの丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１５－１）（０．３ｇ、０．２４ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ジエチルアミン（０．７５ｇ、２．４ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を濃縮し、ジエチルエーテル及びヘキサンによる粉砕によって精製し、黄色の固体として表題化合物を得た（０．２ｇ、８１．１３％）ＬＣＭＳ：１０２４．９ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-aminoacetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-(( di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino )-Synthesis of tert-butyl-5-oxopentanoate (INX-A15-2)

procedure:
In a 25 mL round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((6aR, 6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo- 2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1 ,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-tert-butyl-5-oxopentanoate (INX-A15-1) (0.3g, 0.24mmol) and THF (5 mL). To this solution was added diethylamine (0.75 g, 2.4 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated and purified by trituration with diethyl ether and hexane to give the title compound as a yellow solid (0.2 g, 81.13%) LCMS: 1024.9 [ M+H] ⁺ .

手順：
１０ｍＬのガラスバイアルに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１５－２）（０．２ｇ、０．２０ｍｍｏｌ）及びＤＣＭ（４ｍＬ）を充填した。この溶液に、水（０．３ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０８２ｇ、０．８９ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０８２ｇ、０．４５ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として粗製の表題化合物ＩＮＸ－Ａ１５－３生成物を得た（０．１８ｇ、７８．５９％）ＬＣＭＳ：１１４５．４［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b- (2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptane- Synthesis of tert-butyl (2-yl)amino)-5-oxopentanoate (INX-A15-3)

procedure:
In a 10 mL glass vial, (S)-4-(2-aminoacetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b -(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a, 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptane tert-Butyl-2-yl)amino)-5-oxopentanoate (INX-A15-2) (0.2 g, 0.20 mmol) and DCM (4 mL) were charged. To this solution was added Na ₂ CO ₃ (0.082 g, 0.89 mmol) dissolved in water (0.3 mL) followed by bromoacetyl bromide (0.082 g, 0.45 mmol) at room temperature for 1 h. Stirred. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude title compound INX-A15-3 product as an off-white solid (0.18 g, 78.59% ) LCMS: 1145.4 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１５－３）（０．１８ｇ、０．１７ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＴＦＡ（０．３ｍＬ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させて、粗生成物を得た。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－ＰＡＣＫ ＯＤＳ－ＡＱ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル：メタノール：２－プロパノール（６５：２５：１０）、Ａ：Ｂ、５５：４５、保持時間１５．６０分）によって精製し、白色の固体として表題化合物を得た（０．０２１ｇ、１２．６４％）ＬＣＭＳ：９７６．５［Ｍ＋Ｈ］^＋。^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ－ｄ６， 主要プロトン割り当て）：^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ： δ ７．３１－７．２８（ｍ， ３Ｈ）， ６．７２（ｄ， ２Ｈ）， ６．１６（ｄ， Ｊ＝１０．８Ｈｚ， １Ｈ）， ５．９２（ｓ， １Ｈ）， ５．４１（ｓ， アセタール－Ｈ， １Ｈ）， ４．９５（ｄ， Ｃ１６Ｈ， １Ｈ）， ４，８５－３．５０（ｍ， １０Ｈ）， ２．７２－１．６０（ｍ， ２１Ｈ）， １．３９（ｓ， ３Ｈ）， １．１０－０．９８（ｍ， ２Ｈ）， ０．８８（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H -naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5 -Synthesis of oxopentanoic acid (INX-A15)

procedure:
In a 10 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R , 11aR, 12aS, 12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7 , 8,8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl) Tert-butyl phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A15-3) (0.18 g, 0.17 mmol) and DCM (5 mL) were charged. To this solution was added TFA (0.3 mL) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to obtain the crude product. The crude product was subjected to preparative HPLC (column: YMC-PACK ODS-AQ Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile: methanol: 2- Purification by propanol (65:25:10), A:B, 55:45, retention time 15.60 min) to give the title compound as a white solid (0.021 g, 12.64%) LCMS: 976 .5 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d6, major proton assignment): ¹ H NMR (400 MHz, DMSO: δ 7.31-7.28 (m, 3H), 6.72 (d, 2H), 6. 16 (d, J=10.8Hz, 1H), 5.92 (s, 1H), 5.41 (s, acetal-H, 1H), 4.95 (d, C16H, 1H), 4,85- 3.50 (m, 10H), 2.72-1.60 (m, 21H), 1.39 (s, 3H), 1.10-0.98 (m, 2H), 0.88 (s, 3H).

（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）オキシ）フェニル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－Ａ１６－１）の合成

手順：
５０ｍＬの一つ口丸底フラスコに、（６－（４－ホルミルフェノキシ）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔブチル（ＩＮＸ－ＳＭ－４３－１）（１．１ｇ、３．３２ｍｍｏｌ）、（６Ｓ，８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－６，９－ジフルオロ－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－デカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（ＩＮＸ－Ｓ－１）（１．０９ｇ、２．６５ｍｍｏｌ）及びＤＣＭ（２０ｍＬ）を充填した。この溶液に、ＭｇＳＯ_４（１．９９ｇ、１６．６１ｍｍｏｌ）、続いて、ＨＣｌＯ_４（１．６ｇ、１６．６１ｍｍｏｌ）を添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、薄黄色の固体として表題化合物を得た（１．３ｇ、粗製）ＬＣＭＳ：６２５．９ ［Ｍ＋Ｈ］^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptane- Synthesis of 2-yl)amino)-5-oxopentanoic acid (INX-A16)

(2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)oxy)phenyl)-2, 6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H -Synthesis of naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-A16-1)

procedure:
In a 50 mL single-necked round bottom flask, add tert-butyl (6-(4-formylphenoxy)spiro[3.3]heptan-2-yl)carbamate (INX-SM-43-1) (1.1 g, 3 .32 mmol), (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10, 13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-decahydro-3H-cyclopenta[a]phenanthren-3-one (INX-S-1) (1 .09 g, 2.65 mmol) and DCM (20 mL). To this solution was added MgSO ₄ (1.99 g, 16.61 mmol) followed by HClO ₄ (1.6 g, 16.61 mmol) and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated _NaHCO3 solution and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a pale yellow solid (1.3 g, crude) LCMS: 625.9 [M+H] ⁺

手順：
３５ｍＬのガラスバイアルに、（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）オキシ）フェニル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オンＩＮＸ－Ａ１６－１）（１．２９ｇ、２．０８ｍｍｏｌ）、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（１．０ｇ、２．０８ｍｍｏｌ）及びＤＭＦ（１０ｍＬ）を充填した。この溶液に、ＨＡＴＵ（０．９４ｇ、２．４９ｍｍｏｌ）及びＤＩＰＥＡ（０．８９ｍＬ、５．２０ｍｍｏｌ）を反応混合物に添加し、室温で３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィー（水：アセトニトリル４６：５４）によって精製し、薄黄色の固体として表題化合物を得た（０．８ｇ、３５．３２％）ＬＣＭＳ：１０９１．５ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS,6bR,7S,8aS , 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7 , 8,8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl) Synthesis of tert-butyl phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A16-2)

procedure:
In a 35 mL glass vial, (2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)oxy) ) phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a , 12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one INX-A16-1) (1.29 g, 2. 08 mmol), (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentanoic acid (INX-P -4) (1.0 g, 2.08 mmol) and DMF (10 mL) were charged. To this solution, HATU (0.94 g, 2.49 mmol) and DIPEA (0.89 mL, 5.20 mmol) were added to the reaction mixture and stirred at room temperature for 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography (water:acetonitrile 46:54) to give the title compound as a pale yellow solid (0.8 g, 35.32%) LCMS: 1091.5 [M+H] ⁺ .

手順：
３５ｍＬのガラスバイアルに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１６－２）（０．３８ｇ、０．３２ｍｍｏｌ）及びＤＭＦ（１．５ｍＬ）を添加した。この溶液に、１Ｈ－テトラゾール（０．２４ｇ、３．４０ｍｍｏｌ）及び（ｔＢｕＯ）_２ＰＮ（ｉ－Ｐｒ）_２（２．３ｇ、８．２１ｍｍｏｌ）を添加し、室温で１６時間撹拌した。反応混合物を０℃で冷却し、過酸化水素（４ｍＬ）を反応混合物中に添加し、室温で１時間撹拌した。反応混合物を逆相カラムクロマトグラフィー（アセトニトリル：水、８０：２０）にかけて、オフホワイト色の固体として表題化合物を得た（０．２ｇ、４８．７３％）ＬＣＭＳ：１２８３．７ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS,6bR,7S,8aS ,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ Synthesis of tert-butyl 1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A16-3)

procedure:
In a 35 mL glass vial, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((2S,6aS) , 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4 , 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3] tert-butyl dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A16-2) (0.38 g, 0.32 mmol) and DMF ( 1.5 mL) was added. To this solution were added 1H-tetrazole (0.24 g, 3.40 mmol) and (tBuO) ₂ PN(i-Pr) ₂ (2.3 g, 8.21 mmol) and stirred at room temperature for 16 hours. The reaction mixture was cooled at 0° C., hydrogen peroxide (4 mL) was added into the reaction mixture and stirred at room temperature for 1 hour. The reaction mixture was subjected to reverse phase column chromatography (acetonitrile:water, 80:20) to give the title compound as an off-white solid (0.2 g, 48.73%) LCMS: 1283.7 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１６－３）（０．１７ｇ、０．１３ｍｍｏｌ）及びＴＨＦ（３ｍＬ）を充填した。この溶液に、ジエチルアミン（０．０９６ｇ、１．３ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で濃縮した。粗製物をジエチルエーテル及びヘキサンで粉砕し、黄色の固体として表題化合物を得た（０．１３ｇ、９４．３２％）。ＬＣＭＳ：１０６０．８［Ｍ＋Ｈ］＋ (S)-4-(2-aminoacetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b-(2- ((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3 ] Synthesis of tert-butyl heptane-2-yl)amino)-5-oxopentanoate (INX-A16-4)

procedure:
In a 25 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-( (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy -6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5 tert-butyl indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A16- 3) (0.17 g, 0.13 mmol) and THF (3 mL) were charged. Diethylamine (0.096 g, 1.3 mmol) was added to this solution and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated under vacuum. The crude was triturated with diethyl ether and hexane to give the title compound as a yellow solid (0.13 g, 94.32%). LCMS: 1060.8 [M+H]+

手順：
１０ｍＬのガラスバイアルに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１６－４）（０．１３ｇ、０．１２ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、水（０．３ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０５１ｇ、０．４９ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０４９ｇ、０．２４ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として粗製の表題化合物ＩＮＸ－Ａ１６－５生成物を得た（０．１２ｇ、８４．６６％）。ＬＣＭＳ １１８２．１ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8 , 8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy) Synthesis of tert-butyl spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A16-5)

procedure:
In a 10 mL glass vial, (S)-4-(2-aminoacetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) -8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7, 8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy ) tert-butyl spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A16-4) (0.13 g, 0.12 mmol) and DCM (3 mL) were charged. To this solution was added Na ₂ CO ₃ (0.051 g, 0.49 mmol) dissolved in water (0.3 mL) followed by bromoacetyl bromide (0.049 g, 0.24 mmol) for 1 h at room temperature. Stirred. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude title compound INX-A16-5 product as an off-white solid (0.12 g, 84.66% ). LCMS 1182.1 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）フェノキシ）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１６－５）（０．１２ｇ、０．１０ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．０５７ｇ、０．５０ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ（２５０×１９）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝６４：３６、保持時間１２．８６分）によって精製し、白色の固体として表題化合物を得た（０．０１３ｇ、１２．８３％）ＬＣＭＳ：１０１２．４［Ｍ＋Ｈ］^＋。^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ： δ：８．５４（ｔ， １Ｈ）， ８．１１－８．０９（ｍ， ２Ｈ）， ７．３３－７．２５（ｍ， ３Ｈ）， ６．８４（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．３０（ｄｄ， Ｊ＝１０．４ ＆ １．６Ｈｚ， １Ｈ）， ６．１３（ｓ， １Ｈ）， ５．７２－５．６０（ｍ， ＣＨ－Ｆ， １Ｈ）， ５．４８（ｓ， アセタール－Ｈ， １Ｈ）， ４．９３（ｄ， Ｊ＝４．８Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．９０－４．９１（ｍ， １Ｈ）， ４．６１－４．５７（ｍ， ２Ｈ）， ４．３０－４．００（ｍ， ５Ｈ）， ３．９４（ｓ， ２Ｈ）， ３．００－１．６７（ｍ， ２０Ｈ）， １．５０－１．４８（ｍ， ３Ｈ）， ０．８９（ｓ， ３Ｈ） (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)- 2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)spiro[3.3]heptane- Synthesis of 2-yl)amino)-5-oxopentanoic acid (INX-A16)

procedure:
In a 10 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS) ,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2 ,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1, 3] tert-butyl dioxol-10-yl)phenoxy)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoate (INX-A16-5) (0.12 g, 0.10 mmol) and DCM (2 mL) was charged. To this solution was added TFA (0.057 g, 0.50 mmol) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: SUNFIRE Prep C18 OBD (250 x 19) mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 64:36, retention time 12 .86 min) to give the title compound as a white solid (0.013 g, 12.83%) LCMS: 1012.4 [M+H] ⁺ . ^1H NMR (400 MHz, DMSO: δ: 8.54 (t, 1H), 8.11-8.09 (m, 2H), 7.33-7.25 (m, 3H), 6.84 ( d, J=8.4Hz, 2H), 6.30(dd, J=10.4 & 1.6Hz, 1H), 6.13(s, 1H), 5.72-5.60(m, CH -F, 1H), 5.48 (s, acetal-H, 1H), 4.93 (d, J=4.8Hz, C16H, 1H), 4.90-4.91 (m, 1H), 4 .61-4.57 (m, 2H), 4.30-4.00 (m, 5H), 3.94 (s, 2H), 3.00-1.67 (m, 20H), 1.50 -1.48 (m, 3H), 0.89 (s, 3H)

ステップ１：（Ｅ）－６－（（２－トシルヒドラゾノ）メチル）－２－アザスピロ［３．３］ヘプタン－２－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４７－１）の合成

手順：
３５ｍＬのガラスバイアルに、窒素下で６－ホルミル－２－アザスピロ［３．３］ヘプタン－２－カルボン酸ｔｅｒｔ－ブチル（０．２ｇ、０．８８ｍｍｏｌ）及びＥｔＯＨ（１０ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホンヒドラジド（０．２４７ｇ、１．３３３ｍｍｏｌ）及び触媒量のＡｃＯＨ（０．１ｍＬ）を添加し、室温で０．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、固体を濾過し、真空下で乾燥させて、白色の固体として表題化合物を得た（０．３ｇ、８６．６％）。ＬＣＭＳ：３９４．２ ［Ｍ＋Ｈ］^＋ (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((2-Azaspiro[3.3]heptan-6-yl)methyl)phenyl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-47)

Step 1: Synthesis of tert-butyl (E)-6-((2-tosylhydrazono)methyl)-2-azaspiro[3.3]heptane-2-carboxylate (INX-SM-47-1)

procedure:
A 35 mL glass vial was charged with tert-butyl 6-formyl-2-azaspiro[3.3]heptane-2-carboxylate (0.2 g, 0.88 mmol) and EtOH (10 mL) under nitrogen. To this solution was added p-toluenesulfone hydrazide (0.247 g, 1.333 mmol) and a catalytic amount of AcOH (0.1 mL) and stirred at room temperature for 0.5 h. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the solid was filtered and dried under vacuum to give the title compound as a white solid (0.3 g, 86.6%). LCMS: 394.2 [M+H] ⁺

手順：
３５ｍＬのガラスバイアルに、窒素下で（Ｅ）－６－（（２－トシルヒドラゾノ）メチル）－２－アザスピロ［３．３］ヘプタン－２－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４７－１）（０．３ｇ、０．７６２ｍｍｏｌ）及びジオキサン（５ｍＬ）を充填した。この溶液に、（４－ホルミルフェニル）ボロン酸（０．１７１ｇ、１．１４３ｍｍｏｌ）及びＫ_２ＣＯ_３（０．１５８ｇ、１．１４３ｍｍｏｌ）を添加し、１００℃で３時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、５０：５０）によって精製し、黄色の粘着性固体として表題化合物を得た（０．０６５ｇ、２７．０３％）。ＬＣＭＳ：３１６．２ ［Ｍ＋Ｈ］^＋ Synthesis of tert-butyl 6-(4-formylbenzyl)-2-azaspiro[3.3]heptane-2-carboxylate (INX-SM-47-2)

procedure:
In a 35 mL glass vial, tert-butyl (E)-6-((2-tosylhydrazono)methyl)-2-azaspiro[3.3]heptane-2-carboxylate (INX-SM-47-1) was added under nitrogen. (0.3 g, 0.762 mmol) and dioxane (5 mL). To this solution was added (4-formylphenyl)boronic acid (0.171 g, 1.143 mmol) and K ₂ CO ₃ (0.158 g, 1.143 mmol) and stirred at 100° C. for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 50:50) to give the title compound as a yellow sticky solid (0.065 g, 27.03%). LCMS: 316.2 [M+H] ⁺

手順：
２５ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の６－（４－ホルミルベンジル）－２－アザスピロ［３．３］ヘプタン－２－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４７－２）（０．０６５ｇ、０．２０６ｍｍｏｌ）及び（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．０７７ｇ、０．２０６ｍｍｏｌ）を充填した。この溶液に、ＭｇＳＯ_４（０．１０３ｇ、１．０３ｍｍｏｌ）及びＨＣｌＯ４（０．１２３ｇ、１．０３ｍｍｏｌ）を添加し、室温で更に１．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、真空下で濃縮した。粗製物を冷水で粉砕し、固体を濾過した。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－ＰＡＣＫ ＯＤＳ－ＡＱ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル：メタノール：２－プロパノール（６５：２５：１０）、Ａ：Ｂ、５８：４２、保持時間１２．２３分）によって精製し、白色の固体として表題化合物を得た（０．０２５ｇ、１７．７７％）。ＬＣＭＳ：５７４．４ ［Ｍ＋Ｈ］^＋ ；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ： δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３７（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．１８（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０ ＆ ２．０Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４６（ｓ， アセタール－Ｈ， １Ｈ）， ５．０６（ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．６５－４．６０（ｍ， ２Ｈ）， ４．４４（ｄ， Ｊ＝２．８Ｈｚ， １Ｈ）， ４．３５－４．３１（ｍ， １Ｈ）， ４．０３（ｓ， ２Ｈ）， ３．９５（ｓ， ２Ｈ）， ２．７０－１．６０（ｍ， １５Ｈ）， １．５０（ｓ， ３Ｈ）， １．１７－１．０４（ｍ， ２Ｈ）， １．０１（ｓ， ３Ｈ） (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((2-Azaspiro[3.3]heptan-6-yl)methyl)phenyl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetic acid (INX-SM-47)

procedure:
In a 25 mL single neck round bottom flask was added tert-butyl 6-(4-formylbenzyl)-2-azaspiro[3.3]heptane-2-carboxylate (INX-SM-47-2) in DCM (2 mL). ) (0.065g, 0.206mmol) and (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13 -dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0 0.077g, 0.206mmol). To this solution were added _MgSO4 (0.103g, 1.03mmol) and HClO4 (0.123g, 1.03mmol) and stirred for an additional 1.5 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated _NaHCO3 solution and concentrated under vacuum. The crude was triturated with cold water and the solids were filtered. The crude product was subjected to preparative HPLC (column: YMC-PACK ODS-AQ Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile: methanol: 2- Purification by propanol (65:25:10), A:B, 58:42, retention time 12.23 min) gave the title compound as a white solid (0.025 g, 17.77%). LCMS: 574.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD: δ: 7.47 (d, J = 10.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H) , 7.18 (d, J=8.4Hz, 2H), 6.27 (dd, J=10.0 & 2.0Hz, 1H), 6.04 (s, 1H), 5.46 (s, Acetal-H, 1H), 5.06 (d, J=5.2Hz, C16H, 1H), 4.65-4.60 (m, 2H), 4.44 (d, J=2.8Hz, 1H) ), 4.35-4.31(m, 1H), 4.03(s, 2H), 3.95(s, 2H), 2.70-1.60(m, 15H), 1.50( s, 3H), 1.17-1.04(m, 2H), 1.01(s, 3H)

３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）シクロブタン－１－カルボン酸カルバミン酸メチル（ＩＮＸ－ＳＭ－４９－１）の合成

手順：
１００ｍＬの一つ口丸底フラスコに、３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）シクロブタン－１－カルボン酸（３．０ｇ、１３．９５ｍｍｏｌ）、炭酸カリウム（３．８ｇ、２７．９０ｍｍｏｌ）、及びＤＭＦ（２０ｍＬ）を充填した。この溶液に、ヨウ化メチル（２．９ｇ、２０．９２ｍｍｏｌ）を添加し、室温で更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＨ_２Ｏで希釈し、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、黄色の固体として表題化合物を得た（２．２ｇ、６８．８５％）。ＬＣＭＳ：２３０．２０ ［Ｍ＋Ｈ］^＋ (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-10-(4-((3-(methyl (amino)cyclobutyl)methyl)phenyl)-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno Synthesis of [1,2-d][1,3]dioxol-4-one (INX-SM-49)

Synthesis of 3-((tert-butoxycarbonyl)amino)cyclobutane-1-carboxylic acid methyl carbamate (INX-SM-49-1)

procedure:
In a 100 mL single-neck round bottom flask, 3-((tert-butoxycarbonyl)amino)cyclobutane-1-carboxylic acid (3.0 g, 13.95 mmol), potassium carbonate (3.8 g, 27.90 mmol), and DMF (20 mL) was charged. To this solution was added methyl iodide (2.9 g, 20.92 mmol) and stirred for an additional hour at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with H ₂ O and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a yellow solid (2.2 g, 68.85%). LCMS: 230.20 [M+H] ⁺

手順：
１００ｍＬの一つ口丸底フラスコに、ＤＭＦ（２０ｍＬ）中の３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）シクロブタン－１－カルボン酸カルバミン酸メチル（ＩＮＸ－ＳＭ－４９－１）（２．２ｇ、９．６０ｍｍｏｌ）及び６０％水素化ナトリウム（０．７７ｇ、１９．２１ｍｍｏｌ）を０℃で充填した。この溶液に、ヨウ化メチル（２．０３ｇ、１４．４０ｍｍｏｌ）を添加し、室温で更に１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、黄色の固体として表題化合物を得た（１．８ｇ、７７．０％）。ＬＣＭＳ：２４４．２０ ［Ｍ＋Ｈ］^＋ Synthesis of methyl 3-((tert-butoxycarbonyl)(methyl)amino)cyclobutane-1-carboxylate (INX-SM-49-2)

procedure:
In a 100 mL single neck round bottom flask, add methyl 3-((tert-butoxycarbonyl)amino)cyclobutane-1-carboxylic acid carbamate (INX-SM-49-1) (2.2 g, 9.60 mmol) and 60% sodium hydride (0.77 g, 19.21 mmol) at 0°C. To this solution was added methyl iodide (2.03 g, 14.40 mmol) and stirred for an additional 16 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a yellow solid (1.8 g, 77.0%). LCMS: 244.20 [M+H] ⁺

手順：
１００ｍＬの一つ口丸底フラスコに、ＴＨＦ：ＭｅＯＨ（１：１、２０ｍＬ）中の３－（（ｔｅｒｔ－ブトキシカルボニル）（メチル）アミノ）シクロブタン－１－カルボン酸メチル（ＩＮＸ－ＳＭ－４９－２）（１．８ｇ、７．４０ｍｍｏｌ）を充填し、水素化ホウ素ナトリウム（１．３ｇ、３７．０ｍｍｏｌ）を添加し、室温で更に５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水で希釈し、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、ゴム状の固体として表題化合物を得た（１．７ｇ、粗製）。ＬＣＭＳ：２１６．１［Ｍ＋Ｈ］^＋ Synthesis of tert-butyl (3-(hydroxymethyl)cyclobutyl)(methyl)carbamate (INX-SM-49-3)

procedure:
In a 100 mL single neck round bottom flask, add methyl 3-((tert-butoxycarbonyl)(methyl)amino)cyclobutane-1-carboxylate (INX-SM-49-) in THF:MeOH (1:1, 20 mL). 2) (1.8 g, 7.40 mmol), sodium borohydride (1.3 g, 37.0 mmol) was added, and the mixture was further stirred at room temperature for 5 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a gummy solid (1.7 g, crude). LCMS: 216.1 [M+H] ⁺

手順：
１００ｍＬの一つ口丸底フラスコに、ＤＣＭ（２０ｍＬ）中の（３－（ヒドロキシメチル）シクロブチル）（メチル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４９－３）（１．７ｇ、粗製）を充填した。この溶液に、ＤＭＰ（５．０２ｇ、１１．８０ｍｍｏｌ）を室温で添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、ジクロロメタンで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、ゴム状の固体として表題化合物を得た（１．５ｇ、８９．０６％）。粗製物を即時に次のステップに使用した。 Synthesis of tert-butyl (3-formylcyclobutyl)(methyl)carbamate (INX-SM-49-4)

procedure:
In a 100 mL single neck round bottom flask, add tert-butyl (3-(hydroxymethyl)cyclobutyl)(methyl)carbamate (INX-SM-49-3) (1.7 g, crude) in DCM (20 mL). Filled. DMP (5.02 g, 11.80 mmol) was added to this solution at room temperature and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated NaHCO ₃ solution and extracted with dichloromethane. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a gummy solid (1.5 g, 89.06%). The crude material was used immediately in the next step.

手順：
１０ｍＬのガラスバイアルに、（３－ホルミルシクロブチル）（メチル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４９－４）（１．５ｇ、７．０４ｍｍｏｌ）及びエタノール（２０ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホニルヒドラジド（１．４ｇ、７．７ｍｍｏｌ）及び酢酸（触媒）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、結果として得られた固体を濾過し、真空下で乾燥させて、白色の固体として表題化合物を得た（２．０ｇ、７４．４６％）。ＬＣＭＳ：３８２．２２ ［Ｍ＋Ｈ］^＋ Synthesis of tert-butyl (E)-methyl(3-((2-tosylhydrazono)methyl)cyclobutyl)carbamate (INX-SM-49-5)

procedure:
A 10 mL glass vial was charged with tert-butyl (3-formylcyclobutyl)(methyl)carbamate (INX-SM-49-4) (1.5 g, 7.04 mmol) and ethanol (20 mL). To this solution, p-toluenesulfonyl hydrazide (1.4 g, 7.7 mmol) and acetic acid (catalyst) were added and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the resulting solid was filtered and dried under vacuum to give the title compound as a white solid (2.0 g, 74.46 %). LCMS: 382.22 [M+H] ⁺

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｅ）－メチル（３－（（２－トシルヒドラゾノ）メチル）シクロブチル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４９－５）（２．０ｇ、５．２４ｍｍｏｌ）及びジオキサン（２０ｍＬ）を充填した。この溶液に、（４－ホルミルフェニル）ボロン酸（１．１７ｇ、７．８７ｍｍｏｌ）及びＫ_２ＣＯ_３（１．０８ｇ、７．８７ｍｍｏｌ）を添加し、１１０℃で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、１：１）によって精製し、無色の液体として表題化合物を得た（０．５００ｇ、３１．４５％）。ＬＣＭＳ：３０４．２ ［［Ｍ＋Ｈ］^＋ Synthesis of tert-butyl (3-(4-formylbenzyl)cyclobutyl)(methyl)carbamate (INX-SM-49-6)

procedure:
In a 50 mL single neck round bottom flask, add tert-butyl (E)-methyl(3-((2-tosylhydrazono)methyl)cyclobutyl)carbamate (INX-SM-49-5) (2.0 g, 5.24 mmol). ) and dioxane (20 mL). To this solution was added (4-formylphenyl)boronic acid (1.17 g, 7.87 mmol) and K ₂ CO ₃ (1.08 g, 7.87 mmol) and stirred at 110° C. for an additional 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 1:1) to give the title compound as a colorless liquid (0.500 g, 31.45%). LCMS: 304.2 [[M+H] ⁺

手順：
２５ｍＬの一つ口丸底フラスコに、（３－（４－ホルミルベンジル）シクロブチル）（メチル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４９－６）（０．１６ｇ、０．５２ｍｍｏｌ）、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．２１ｇ、０．５８ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＭｇＳＯ_４（０．３１ｇ、２．６ｍｍｏｌ）及びＨＣｌＯ_４（０．２６ｇ、２．６ｍｍｏｌ）を添加し、室温で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、ＭＤＣで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物を分取ＨＰＬＣによって精製し、白色の固体として表題化合物を得た（０．０５０ｇ、１７．１１％）。ＬＣＭＳ：５６２．４［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ－ｄ６－ｄ２Ｏ： δ：７．３７（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ７．３２（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．１７（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．１７（ｄｄ， Ｊ＝１０ ＆ １．６Ｈｚ， １Ｈ）， ５．９４（ｓ， １Ｈ）， ５．４１（ｓ， アセタール－Ｈ， １Ｈ）， ４．９３（ｄ， Ｊ＝４．８Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．４８（ｍ， １Ｈ）， ４．３１－４．２９（ｍ， １Ｈ）， ４．２１－４．１５（ｍ， １Ｈ）， ３．５０－３．４０（ｍ， １Ｈ）， ２．６９－１．６０（ｍ， １９Ｈ）， １．４０（ｓ， ３Ｈ）， ０．９９－０．９６（ｍ， ２Ｈ）， ０．８７（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-10-(4-((3-(methyl (amino)cyclobutyl)methyl)phenyl)-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno Synthesis of [1,2-d][1,3]dioxol-4-one (INX-SM-49)

procedure:
In a 25 mL single-necked round bottom flask, add tert-butyl (3-(4-formylbenzyl)cyclobutyl)(methyl)carbamate (INX-SM-49-6) (0.16 g, 0.52 mmol), (8S ,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10, 11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-α-hydroxyprednisolone) (0.21 g, 0.58 mmol) and DCM (2 mL). Filled. To this solution were added MgSO ₄ (0.31 g, 2.6 mmol) and HClO ₄ (0.26 g, 2.6 mmol) and stirred for an additional 2 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated _NaHCO3 solution and extracted with MDC. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the crude product. The crude material was purified by preparative HPLC to give the title compound as a white solid (0.050 g, 17.11%). LCMS: 562.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, DMSO-d6-d2O: δ: 7.37 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 10. 0Hz, 1H), 7.17 (d, J=8.0Hz, 2H), 6.17 (dd, J=10 & 1.6Hz, 1H), 5.94 (s, 1H), 5.41 ( s, acetal-H, 1H), 4.93 (d, J=4.8Hz, C16H, 1H), 4.48 (m, 1H), 4.31-4.29 (m, 1H), 4. 21-4.15 (m, 1H), 3.50-3.40 (m, 1H), 2.69-1.60 (m, 19H), 1.40 (s, 3H), 0.99- 0.96 (m, 2H), 0.87 (s, 3H).

（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（（２Ｓ，３Ｒ，４Ｒ，５Ｓ）－４－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）キュバン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２－１）の合成

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）０．１３７ｇ、０．２８５ｍｍｏｌ）、ＨＡＴＵ（０．１６３ｇ、０．４２８ｍｍｏｌ）、及びＤＭＦ（２ｍＬ）を充填した。この溶液に、ＤＩＰＥＡ（０．１１０ｇ、０．８５７ｍｍｏｌ）及び（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（（１ｓ，２Ｒ，３Ｓ，８Ｓ）－４－アミノキュバン－１－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－９）（０．１６０ｇ、０．０．２８５ｍｍｏｌ）を添加し、反応混合物を室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を逆相カラムクロマトグラフィーによって精製し、淡黄色の固体として表題化合物を得た（０．０８ｇ、２６．３９％）。ＬＣＭＳ：１０６０．７［Ｍ＋Ｈ］^＋ (4S)-4-(2-(2-bromoacetamide)acetamide)-5-(((2S,3R,4R,5S)-4-(4-((6aR,6bS,7S,8aS,8bS,10R , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cuban-1-yl)amino) Synthesis of -5-oxopentanoic acid (INX-A2)

(4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(((2S,3R,4R,5S)-4-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- Synthesis of tert-butyl)benzyl)cuban-1-yl)amino)-5-oxopentanoate (INX-A2-1)

procedure:
In a 25 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.137 g, 0.285 mmol), HATU (0.163 g, 0.428 mmol), and DMF (2 mL) were charged. To this solution was added DIPEA (0.110 g, 0.857 mmol) and (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-(((1s, 2R, 3S, 8S)- 4-aminocuban-1-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a , 12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-9) (0 .160 g, 0.0.285 mmol) was added and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by reverse phase column chromatography to give the title compound as a pale yellow solid (0.08 g, 26.39%). LCMS: 1060.7 [M+H] ⁺

手順：
２５ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（（２Ｓ，３Ｒ，４Ｒ，５Ｓ）－４－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）キュバン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２－１）（０．０８ｇ、０．０７５ｍｍｏｌ）及びＴＨＦ（３ｍＬ）を充填した。この溶液に、ジエチルアミン（０．０５５ｇ、０．７５ｍｍｏｌ）及び１，８－ジアザビシクロ（５．４．０）ウンデス－７－エン（ＤＢＵ）（触媒）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で濃縮した。粗製物をＤＣＭ及びヘキサンで粉砕し、白色の固体として表題化合物を得た（０．０６ｇ、９５．４６％）。ＬＣＭＳ：８３９．４ ［Ｍ＋Ｈ］^＋。 (4S)-4-(2-aminoacetamide)-5-(((2S,3R,4R,5S)-4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cuban-1-yl)amino)-5-oxopentane Synthesis of tert-butyl acid (INX-A2-2)

procedure:
In a 25 mL single neck round bottom flask, add (4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(((2S,3R,4R ,5S)-4-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ tert-butyl 1,3]dioxol-10-yl)benzyl)cuban-1-yl)amino)-5-oxopentanoate (INX-A2-1) (0.08 g, 0.075 mmol) and THF (3 mL) filled with. To this solution were added diethylamine (0.055 g, 0.75 mmol) and 1,8-diazabicyclo(5.4.0) undec-7-ene (DBU) (catalyst) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated under vacuum. The crude was triturated with DCM and hexane to give the title compound as a white solid (0.06g, 95.46%). LCMS: 839.4 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（（２Ｓ，３Ｒ，４Ｒ，５Ｓ）－４－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）キュバン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２－２）（０．０９ｇ、０．１０７ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、水（０．１ｍＬ）に溶解したＮａ_２ＣＯ_３（０．０２２ｇ、０．２１ｍｍｏｌ）、続いて、ブロモアセチルブロミド（０．０４３ｇ、０．０２１ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（０．０６５ｇ、６３．１１％）。
ＬＣＭＳ：Ｃ５０Ｈ６１^７９ＢｒＮ_３Ｏ_１１に対する計算値（９５８．３５）、実測値９５８．２［Ｍ＋Ｈ］^＋。 (4S)-4-(2-(2-bromoacetamide)acetamide)-5-(((2S,3R,4R,5S)-4-(4-((6aR,6bS,7S,8aS,8bS,10R , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cuban-1-yl)amino) Synthesis of tert-butyl-5-oxopentanoate (INX-A2-3)

procedure:
In a 25 mL single neck round bottom flask, add (4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(((2S,3R,4R ,5S)-4-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ tert-butyl 1,3]dioxol-10-yl)benzyl)cuban-1-yl)amino)-5-oxopentanoate (INX-A2-2) (0.09 g, 0.107 mmol) and DCM (2 mL) filled with. To this solution was added Na ₂ CO ₃ (0.022 g, 0.21 mmol) dissolved in water (0.1 mL) followed by bromoacetyl bromide (0.043 g, 0.021 mmol) at room temperature for 1 h. Stirred. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as an off-white solid (0.065 g, 63.11%).
LCMS: Calcd for C50H61 ⁷⁹ BrN ₃ O ₁₁ (958.35), found 958.2 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（（２Ｓ，３Ｒ，４Ｒ，５Ｓ）－４－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）キュバン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２－３）（０．０６５ｇ、０．０６７ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、ＴＦＡ（０．５ｍＬ）及びトリイソプロピル生理食塩水（触媒）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させて、オフホワイト色の固体として表題化合物生成物を得た（０．０５５ｇの粗製物）。ＬＣＭＳ ９０３．５ ［Ｍ＋Ｈ］^＋。 (4S)-4-(2-(2-bromoacetamide)acetamide)-5-(((2S,3R,4R,5S)-4-(4-((6aR,6bS,7S,8aS,8bS,10R , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cuban-1-yl)amino) Synthesis of -5-oxopentanoic acid (INX-A2)

procedure:
In a 25 mL single neck round bottom flask, add (4S)-4-(2-(2-bromoacetamido)acetamide)-5-(((2S,3R,4R,5S)-4-(4-((6aR , 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8 , 8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl) Tert-butyl (cuban-1-yl)amino)-5-oxopentanoate (INX-A2-3) (0.065 g, 0.067 mmol) and DCM (3 mL) were charged. To this solution were added TFA (0.5 mL) and triisopropyl saline (catalyst) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to give the title compound product as an off-white solid (0.055 g crude). LCMS 903.5 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－ＣＹＳ）（０．１ｇ、０．１１ｍｍｏｌ）及びＤＭＦ（１ｍＬ）を充填した。この溶液に、Ｌ－システイン（０．００３ｇ、０．１２ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＬＣＭＳによって示される反応の完了後、反応混合物を凍結乾燥させ、粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、４５：５５）によって精製し、オフホワイト色の固体として表題化合物を得た（０．０５０ｇ、５０．１１％）。ＬＣＭＳ：９０７．５ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ： δ：７．４７（ｄ， Ｊ＝１０．４Ｈｚ， １Ｈ）， ７．３８（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．１４（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２８（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ６．０５（ｓ， １Ｈ）， ５．４６（ｓ， アセタール－Ｈ， １Ｈ）， ５．０６（ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．６５（ｄ， １Ｈ）， ４．４６（ｍ， １Ｈ）， ４．３５ （ｄ， １Ｈ）， ４．１８－４．１６（ｍ， １Ｈ）， ３．９０（ｍ， ２Ｈ）， ３．７９（ｍ， １Ｈ）， ３．５０－３．００ （ｍ， ４Ｈ）， ３．００－１．６０ （ｍ， ２１Ｈ）， １．５２（ｓ， ３Ｈ）， １．３０－１．０７（ｍ， ２Ｈ）， １．０１（ｓ， ３Ｈ）。 (S)-4-(2-(2-(((R)-2-amino-2-carboxyethyl)thio)acetamido)acetamido)-5-((3-(4-((6aR,6bS,7S , 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a ,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[ 1.1.1] Synthesis of pentan-1-yl)amino)-5-oxopentanoic acid (INX-P-CYS)

procedure:
In a 10 mL single neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R) , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1.1] Charged with pentan-1-yl)amino)-5-oxopentanoic acid (INX-P-CYS) (0.1 g, 0.11 mmol) and DMF (1 mL). To this solution was added L-cysteine (0.003 g, 0.12 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by LCMS, the reaction mixture was lyophilized and the crude was purified by reverse phase column chromatography (acetonitrile:water, 45:55) to give the title compound as an off-white solid (0 .050g, 50.11%). LCMS: 907.5 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD: δ: 7.47 (d, J = 10.4 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H) , 7.14(d, J=8.0Hz, 2H), 6.28(d, J=10.0Hz, 1H), 6.05(s, 1H), 5.46(s, Acetal-H, 1H), 5.06 (d, J=5.2Hz, C16H, 1H), 4.65 (d, 1H), 4.46 (m, 1H), 4.35 (d, 1H), 4.18 -4.16 (m, 1H), 3.90 (m, 2H), 3.79 (m, 1H), 3.50-3.00 (m, 4H), 3.00-1.60 (m , 21H), 1.52 (s, 3H), 1.30-1.07 (m, 2H), 1.01 (s, 3H).

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸（ＩＮＸ－Ｖ）（０．１ｇ、０．１１ｍｍｏｌ）及びＤＭＦ（１．５ｍＬ）を充填した。この溶液に、Ｌ－システイン（０．００２７ｇ、０．２２３ｍｍｏｌ）を添加し、室温で１６時間撹拌した。ＬＣＭＳによって示される反応の完了後、反応混合物を凍結乾燥させた。単離した粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝ＭＴＢＥ：アセトニトリル（１０：９０）、Ａ：Ｂ＝７５：２５）、保持時間１２．５分によって精製し、白色の固体として表題化合物を得た（０．０１２ｇ、１０．２４％）。ＬＣＭＳ：９３５．５ ［Ｍ＋Ｈ］^＋；
^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ： δ：７．４６（ｄ， Ｊ＝１０．４Ｈｚ， １Ｈ）， ７．３４（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．１５（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１．４Ｈｚ， １０Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４５（ｓ， アセタール－Ｈ， １Ｈ）， ５．０６（ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ， １Ｈ）， ４．６６（ｄ， １Ｈ）， ４．４５－４．４０（ｍ， １Ｈ）， ４．３６－４．３１（ｍ， ２Ｈ）， ４．１２（ｍ， １Ｈ）， ４．００－３．９２（ｍ， ３Ｈ），
３．５０－１．７３（ｍ， ２８Ｈ）， １．５２（ｓ， ３Ｈ）， １．２０－１．０３（ｍ， ２Ｈ）， １．００（ｓ， ３Ｈ）。 (S)-4-(2-(2-(((R)-2-amino-2-carboxyethyl)thio)acetamido)acetamido)-5-((6 -(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2, 4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3 ] Synthesis of dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoic acid compound (INX-V-CYS)

procedure:
In a 10 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane- 2-yl)amino)-5-oxopentanoic acid (INX-V) (0.1 g, 0.11 mmol) and DMF (1.5 mL) were charged. To this solution was added L-cysteine (0.0027 g, 0.223 mmol) and stirred at room temperature for 16 hours. After completion of the reaction as indicated by LCMS, the reaction mixture was lyophilized. The isolated crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = MTBE: acetonitrile (10 :90), A:B=75:25), retention time 12.5 min to give the title compound as a white solid (0.012 g, 10.24%). LCMS: 935.5 [M+H] ⁺ ;
^1H NMR (400 MHz, MeOD: δ: 7.46 (d, J=10.4Hz, 1H), 7.34 (d, J=8.0Hz, 2H), 7.15 (d, J=8 .0Hz, 2H), 6.27 (dd, J=1.4Hz, 10Hz, 1H), 6.04 (s, 1H), 5.45 (s, acetal-H, 1H), 5.06 (d , J=5.2Hz, C16H, 1H), 4.66 (d, 1H), 4.45-4.40 (m, 1H), 4.36-4.31 (m, 2H), 4.12 (m, 1H), 4.00-3.92 (m, 3H),
3.50-1.73 (m, 28H), 1.52 (s, 3H), 1.20-1.03 (m, 2H), 1.00 (s, 3H).

手順：
１０ｍＬの一つ口丸底フラスコに、（（Ｓ）－６－アミノ－２－（２－（２－ブロモアセトアミド）アセトアミド）－Ｎ－（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）ヘキサンアミド（ＩＮＸ－Ｗ）（０．２ｇ、０．２３ｍｍｏｌ）及びＤＭＦ（１ｍＬ）を充填した。この溶液に、Ｌ－システイン（０．０４１ｇ、０．３４ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＬＣＭＳによって示される反応の完了後、反応混合物を凍結乾燥させた。粗製物を分取ＨＰＬＣ（カラム：ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｓｉｌｉｃａ、ＯＢＤ、１５０－１９ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝７６：２４）、保持時間１９．５分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．０２０ｇ、８．４９％）。ＬＣＭＳ：９０６．４ ［Ｍ＋Ｈ］^＋；
^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ： δ：７．４７（ｄ， Ｊ＝１０．４Ｈｚ， １Ｈ）， ７．３８（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．１４（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２８（ｄ， １Ｈ）， ６．０５（ｓ， １Ｈ）， ５．４８（ｓ， アセタール－Ｈ， １Ｈ）， ５．０７ （ｄ， Ｊ＝５．２Ｈｚ， Ｃ１６Ｈ，１Ｈ），
４．６７（ｄ， １Ｈ）， ４．５０－４．３２（ｍ， ３Ｈ）， ３，３９（ｓ， ２Ｈ）， ３．４０－３．００（ｍ， ２Ｈ）， ３．００－２．９０（ｍ， ２Ｈ）， ２．８５（ｓ， ２Ｈ），
２．７５－１．６０（ｍ， ２２Ｈ）， １．５２（ｓ， ３Ｈ）， １．５０－１．３５（ｍ， ２Ｈ）， １．２０－１．０５（ｍ， ２Ｈ）， １．０１（ｓ， ３Ｈ）。 S-(2-((2-(((S)-6-amino-1-((3-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a , 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1.1. 1] Synthesis of pentan-1-yl)amino)-1-oxohexan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)-L-cysteine compound (INX-W-CYS)

procedure:
In a 10 mL single neck round bottom flask, add ((S)-6-amino-2-(2-(2-bromoacetamido)acetamide)-N-(3-(4-((6aR,6bS,7S,8aS) ,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b , 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)bicyclo[1. 1.1] Pentan-1-yl)hexanamide (INX-W) (0.2 g, 0.23 mmol) and DMF (1 mL) were charged. To this solution was charged L-cysteine (0.041 g, 0.34 mmol). ) was added and stirred for 2 hours at room temperature. After completion of the reaction as indicated by LCMS, the reaction mixture was lyophilized. The crude was subjected to preparative HPLC (column: SUNFIRE Prep Silica, OBD, 150-19 mm, 5 μm; Mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 76:24), retention time 19.5 min) to give the title compound as an off-white solid (0 .020g, 8.49%). LCMS: 906.4 [M+H] ⁺ ;
^1H NMR (400 MHz, MeOD: δ: 7.47 (d, J=10.4Hz, 1H), 7.38 (d, J=8.0Hz, 2H), 7.14 (d, J=8 .0Hz, 2H), 6.28 (d, 1H), 6.05 (s, 1H), 5.48 (s, acetal-H, 1H), 5.07 (d, J=5.2Hz, C16H ,1H),
4.67 (d, 1H), 4.50-4.32 (m, 3H), 3,39 (s, 2H), 3.40-3.00 (m, 2H), 3.00-2. 90 (m, 2H), 2.85 (s, 2H),
2.75-1.60 (m, 22H), 1.52 (s, 3H), 1.50-1.35 (m, 2H), 1.20-1.05 (m, 2H), 1. 01(s, 3H).

スキーム２

スキーム３

２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－ベンジル－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－３４－１）の合成

手順：
１００ｍＬの一つ口丸底フラスコに、（１１β）－２１－（アセチルオキシ）－１１－ヒドロキシプレグナ－１，４，１６－トリエン－３，２０－ジオン（５．０ｇ、１３．０ｍｍｏｌ）及び１，４－ジオキサン（５０ｍＬ）を充填した。この溶液に、Ｎ－（メトキシメチル）－Ｎ－（トリメチルシリルメチル）ベンジルアミン（３０．８ｇ、１３．０ｍｍｏｌ）及びＴＦＡ（０．１０ｇ、０．９ｍｍｏｌ）を添加し、反応混合物を１１０℃で４時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を濃縮した。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル：ヘキサン、２０：８０）によって精製し、オフホワイト色の固体として表題化合物を得た（３．０ｇ、４４．５６％）。ＬＣＭＳ ５１８．２９［Ｍ＋Ｈ］^＋ (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2 ',1':4,5]indeno[1,2-c]pyrrol-4(2H)-one 2,2,2-trifluoroacetate (INX-SM-34) and 2,2,2-trifluoroacetate Contains acetate (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)- 7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2', Synthesis scheme 1 of 1':4,5]indeno[1,2-c]pyrrole-8b(2H)-carboxylic acid compound (INX-SM-42)

Scheme 2

Scheme 3

2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-benzyl-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8 ,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)- Synthesis of 2-oxoethyl acetate (INX-SM-34-1)

procedure:
In a 100 mL single neck round bottom flask, (11β)-21-(acetyloxy)-11-hydroxypregna-1,4,16-triene-3,20-dione (5.0 g, 13.0 mmol) and Charged with 1,4-dioxane (50 mL). To this solution, N-(methoxymethyl)-N-(trimethylsilylmethyl)benzylamine (30.8 g, 13.0 mmol) and TFA (0.10 g, 0.9 mmol) were added and the reaction mixture was heated at 110°C for 4 hours. heated for an hour. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated. The crude material was purified by silica gel column chromatography (ethyl acetate:hexane, 20:80) to give the title compound as an off-white solid (3.0 g, 44.56%). LCMS 518.29 [M+H] ⁺

手順：
１００ｍＬの一つ口丸底フラスコに、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－ベンジル－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－３４－１）（３．０ｇ、５．７９ｍｍｏｌ）及びアセトニトリル（２０ｍＬ）を充填した。この溶液に、ＮａＨＣＯ_３（０．９７ｇ、１１．５ｍｍｏｌ）及びクロロギ酸１－クロロエチル（１．６５ｇ、１１．５９）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライト床を通して濾過した。濾液を真空下で濃縮し、粗製物をｎ－ペンテン及びジエチルエーテルで粉砕して、白色の固体として表題化合物を得た（２．０ｇ、７４．３８％）。ＬＣＭＳ ４２８．３ ［Ｍ＋Ｈ］^＋。 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9 ,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl Synthesis of acetate hydrochloride (INX-SM-34-2)

procedure:
In a 100 mL one-neck round bottom flask, add 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-benzyl-7-hydroxy-6a,8a-dimethyl-4-oxo-1 ,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c] Pyrrole-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-34-1) (3.0 g, 5.79 mmol) and acetonitrile (20 mL) were charged. To this solution was added NaHCO ₃ (0.97 g, 11.5 mmol) and 1-chloroethyl chloroformate (1.65 g, 11.59) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through a bed of Celite. The filtrate was concentrated under vacuum and the crude was triturated with n-pentene and diethyl ether to give the title compound as a white solid (2.0 g, 74.38%). LCMS 428.3 [M+H] ⁺ .

手順：
１０ｍＬのガラスバイアルに、（Ｅ）－（３－（（２－トシルヒドラゾノ）メチル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３－４）（０．０５ｇ、０．１３ｍｍｏｌ）及び１，４－ジオキサン（２ｍＬ）を充填した。この溶液に、Ｋ_２ＣＯ_３（０．０３９ｇ、０．１９ｍｍｏｌ）を添加し、Ｎ_２（ｇ）を１時間パージした。反応混合物中で沈殿を観察した後、（４－ブロモフェニル）ボロン酸（０．０３９ｇ、０．１９ｍｍｏｌ）を反応物に添加し、１１０℃で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、ブラインで洗浄した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：１０：９０）によって精製し、オフホワイト色の固体として表題化合物を得た。（０．０２ｇ、４３．０％）。^１Ｈ ＮＭＲ （ＣＤＣｌ_３） δ：７．３９ （ｄ， Ｊ＝６．４ Ｈｚ， ２Ｈ））， ６．９６ （ｄ， Ｊ＝６．４ Ｈｚ ２Ｈ，）， ２．７９ （ｓ， ２Ｈ）， １．８１（ｓ， ６ Ｈ） １．４４（ｓ， ９Ｈ）。 Synthesis of tert-butyl (3-(4-bromobenzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-34-3)

procedure:
In a 10 mL glass vial, tert-butyl (E)-(3-((2-tosylhydrazono)methyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3-4) ( 0.05 g, 0.13 mmol) and 1,4-dioxane (2 mL). To this solution was added K ₂ CO ₃ (0.039 g, 0.19 mmol) and purged with N _{2 (g)} for 1 hour. After observing precipitation in the reaction mixture, (4-bromophenyl)boronic acid (0.039 g, 0.19 mmol) was added to the reaction and stirred at 110° C. for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and washed with brine. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 10:90) to give the title compound as an off-white solid. (0.02g, 43.0%). ^1H NMR ( _CDCl3 ) δ: 7.39 (d, J=6.4 Hz, 2H)), 6.96 (d, J=6.4 Hz 2H,), 2.79 (s, 2H) , 1.81(s, 6H) 1.44(s, 9H).

手順：
２５ｍＬの一つ口丸底フラスコに、窒素下で（３－（４－ブロモベンジル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３４－３）（０．５ｇ、１．４２ｍｍｏｌ）及び１，４－ジオキサン（１０ｍＬ）を充填した。この溶液に、ビスピナコロンジボラン（ｂｉｓｐｉｎａｃｏｌｏｎｅｄｉｂｏｒａｎｅ）（１．０７ｇ、４．２６ｍｍｏｌ）及び酢酸カリウム（０．２７ｇ、２．８４ｍｍｏｌ）を添加し、Ｎ_２で１５分間パージした。ＰｄＣｌ_２（ｄｐｐｆ）．ＤＣＭ（０．１２ｇ、０．１４ｍｍｏｌ）を反応混合物に添加し、１１０℃で２時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を室温で冷却し、酢酸エチルで希釈した。これをセライト床を通して濾過し、組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：２０：８０）によって精製し、オフホワイト色の半固体として表題化合物を得た（０．８ｇ、粗製）。これを次のステップに使用した。 (3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)carbamic acid tert- Synthesis of butyl (INX-SM-34-4)

procedure:
In a 25 mL single neck round bottom flask, add tert-butyl (3-(4-bromobenzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-34-3) under nitrogen. (0.5 g, 1.42 mmol) and 1,4-dioxane (10 mL). To this solution was added bispinacolonediborane (1.07 g, 4.26 mmol) and potassium acetate (0.27 g, 2.84 mmol) and purged with _N2 for 15 minutes. _PdCl2 (dppf). DCM (0.12g, 0.14mmol) was added to the reaction mixture and heated at 110°C for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was cooled to room temperature and diluted with ethyl acetate. This was filtered through a bed of celite and the combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 20:80) to give the title compound as an off-white semi-solid (0.8 g, crude). This was used in the next step.

手順：
２５ｍＬの一つ口丸底フラスコに、（３－（４－（４，４，５，５－テトラメチル－１，３，２－ジオキサボロラン－２－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチルＩＮＸ－ＳＭ－３４－４）（１．０ｇ、２．５ｍｍｏｌ）及びアセトン：水（９：１）（１０ｍＬ）を充填した。この溶液に、ＮａＩＯ_４（４．２８ｇ、２０．０ｍｍｏｌ）及び酢酸アンモニウム（１．５４ｇ、２０．０ｍｍｏｌ）を添加し、反応混合物を還流温度で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を室温まで冷却し、酢酸エチルで希釈し、セライト床を通して濾過した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をｎ－ペンテン及びジエチルエーテルで粉砕し、オフホワイト色の固体として表題化合物を得た（０．４ｇ、５０．３％）。ＬＣＭＳ：２６２．２ ［Ｍ＋１－ｔ－Ｂｕ］ Synthesis of (4-((3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentan-1-yl)methyl)phenyl)boronic acid (INX-SM-34-5)

procedure:
In a 25 mL single neck round bottom flask, add (3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)bicyclo[1.1.1] tert-Butyl (pentan-1-yl) carbamate INX-SM-34-4) (1.0 g, 2.5 mmol) and acetone:water (9:1) (10 mL) were charged. To this solution, NaIO ₄ (4.28 g, 20.0 mmol) and ammonium acetate (1.54 g, 20.0 mmol) were added and the reaction mixture was stirred at reflux temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was cooled to room temperature, diluted with ethyl acetate, and filtered through a bed of Celite. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was triturated with n-pentene and diethyl ether to give the title compound as an off-white solid (0.4 g, 50.3%). LCMS:262.2 [M+1-t-Bu]

手順：
３５ｍＬのガラスバイアルに、アセトニトリル（５ｍＬ）中の２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート塩酸塩ＩＮＸ－ＳＭ－３４－２）（０．１ｇ、０．２３ｍｍｏｌ）及び（４－（（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）ボロン酸ＩＮＸ－ＳＭ－３４－５）（０．２５ｇ、０．８１ｍｍｏｌ）を充填した。この溶液に、ＫＯＨ（０．１３ｇ、２．３４ｍｍｏｌ）及びＣｕ（ＯＡｃ）_２（０．１２ｇ、０．７０ｍｍｏｌ）を添加し、反応混合物を室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライト床を通して濾過した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：３０：７０）によって精製し、オフホワイト色の固体として表題化合物を得た（０．１５ｇ、９３．３１％）ＬＣＭＳ：６９９．５ ［Ｍ＋Ｈ］^＋。 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-((3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentane-1 -yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b- Synthesis of tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-34-6)

procedure:
In a 35 mL glass vial, 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4) in acetonitrile (5 mL) was added. , 6a, 6b, 7, 8, 8a, 9, 10, 11, 11a, 12, 12a, 12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrole- 8b(2H)-yl)-2-oxoethyl acetate hydrochloride INX-SM-34-2) (0.1 g, 0.23 mmol) and (4-((3-((tert-butoxycarbonyl)amino)bicyclo [1.1.1]Pentan-1-yl)methyl)phenyl)boronic acid INX-SM-34-5) (0.25 g, 0.81 mmol) was charged. To this solution were added KOH (0.13 g, 2.34 mmol) and Cu(OAc) ₂ (0.12 g, 0.70 mmol) and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through a bed of Celite. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was purified by silica gel column chromatography (ethyl acetate/hexane: 30:70) to give the title compound as an off-white solid (0.15 g, 93.31%) LCMS: 699.5 [M+H] ^＋ .

手順：
２５ｍＬの一つ口丸底フラスコに、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－３４－６）（０．１３ｇ、０．１８ｍｍｏｌ）及びメタノール（２ｍＬ）を充填した。この溶液に、Ｋ_２ＣＯ_３（０．０３８ｇ、０．２７ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライト床を通して濾過した。組み合わせた有機層を真空下で蒸発させ、ＤＣＭ（２ｍＬ）に溶解させた。この溶液に、ＴＦＡ（０．２ｍｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝ＭＴＢＥ：アセトニトリル（９０：１０）、Ａ：Ｂ＝７８：２２）によって精製し、ＩＮＸ－ＳＭ－３４を得た（０．０１０ｇ、８．０１％）。ＬＣＭＳ：５５７．３０ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ： δ：７．４９（ｄ， Ｊ＝１０．４Ｈｚ， １Ｈ）， ６．９４（ｄ， Ｊ＝８．８Ｈｚ， ２Ｈ）， ６．６２（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．２６（ｄｄ， Ｊ＝ １０．０ ＆ ２．０ Ｈｚ， １Ｈ）， ５．９９（ｓ， １Ｈ）， ４．６０－４．２５（ｍ， ３Ｈ）， ３．６０－３．００（ｍ， ４Ｈ）， ２．７７－１．５５（ｍ， １８Ｈ）， １．５０（ｓ， ３Ｈ）， １．１５（ｓ， ３Ｈ）， １．１５－１．００（ｍ， ２Ｈ）。
及びＩＮＸ－ＳＭ－４２（０．００４１ｇ、３．３６％）ＬＣＭＳ：５４３．３［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ： δ：７．５０（ｄ， Ｊ＝１０．４Ｈｚ， １Ｈ）， ６．９４（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．６０（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．２６（ｄｄ， Ｊ＝１０．０＆ ２．０Ｈｚ， １Ｈ）， ５．９９（ｓ， １Ｈ）， ４．４５（ｂｒ ｓ， １Ｈ）， ３．６０－３．３０（ｍ， ４Ｈ）， ２．７７（ｓ， ２Ｈ）， ２．７０－１．５５（ｍ， １６Ｈ）， １．５０ （ｓ， ３Ｈ）， １．２２（ｓ， ３Ｈ）， １．１５－１．００（ｍ， ２Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2 ',1':4,5]indeno[1,2-c]pyrrol-4(2H)-one 2,2,2-trifluoroacetate (INX-SM-34) and 2,2,2-trifluoroacetate Contains acetate (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)- 7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2', Synthesis of 1':4,5]indeno[1,2-c]pyrrole-8b(2H)-carboxylic acid compound (INX-SM-42)

procedure:
In a 25 mL single neck round bottom flask, add 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-((3-((tert-butoxycarbonyl)amino)bicyclo [1.1.1] Pentan-1-yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10, 11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX -SM-34-6) (0.13 g, 0.18 mmol) and methanol (2 mL). To this solution was added K ₂ CO ₃ (0.038 g, 0.27 mmol) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through a bed of Celite. The combined organic layers were evaporated under vacuum and dissolved in DCM (2 mL). To this solution was added TFA (0.2 ml) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = MTBE:acetonitrile (90:10) , A:B=78:22) to obtain INX-SM-34 (0.010 g, 8.01%). LCMS: 557.30 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD: δ: 7.49 (d, J = 10.4 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H) , 6.62 (d, J = 8.4Hz, 2H), 6.26 (dd, J = 10.0 & 2.0 Hz, 1H), 5.99 (s, 1H), 4.60-4 .25 (m, 3H), 3.60-3.00 (m, 4H), 2.77-1.55 (m, 18H), 1.50 (s, 3H), 1.15 (s, 3H) ), 1.15-1.00 (m, 2H).
and INX-SM-42 (0.0041 g, 3.36%) LCMS: 543.3 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD: δ: 7.50 (d, J = 10.4 Hz, 1H ), 6.94 (d, J=8.4Hz, 2H), 6.60 (d, J=8.4Hz, 2H), 6.26 (dd, J=10.0&2.0Hz, 1H), 5.99 (s, 1H), 4.45 (br s, 1H), 3.60-3.30 (m, 4H), 2.77 (s, 2H), 2.70-1.55 (m , 16H), 1.50 (s, 3H), 1.22 (s, 3H), 1.15-1.00 (m, 2H).

２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート２，２，２－トリフルオロアセテート（ＩＮＸ－Ａ１３－１）の合成

手順：
１０ｍＬの一つ口丸底フラスコに、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－３４－６）（０．３３ｇ、０．４７ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＴＦＡ（０．１ｍＬ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物をジエチルエーテルで粉砕し、オフホワイト色の固体として表題化合物を得た（０．２８ｇ、定量的）。ＬＣＭＳ：５９９．５ ［Ｍ＋Ｈ］^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydro naphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5- Synthesis of oxopentanoic acid (INX-A13)

2-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)- 7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2', Synthesis of 1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate 2,2,2-trifluoroacetate (INX-A13-1)

procedure:
In a 10 mL single-necked round bottom flask, add 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-((3-((tert-butoxycarbonyl)amino)bicyclo [1.1.1] Pentan-1-yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10, 11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX -SM-34-6) (0.33 g, 0.47 mmol) and DCM (5 mL). To this solution was added TFA (0.1 mL) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude was triturated with diethyl ether to give the title compound as an off-white solid (0.28 g, quantitative). LCMS:599.5 [M+H] ⁺

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．２４ｇ、０．５０ｍｍｏｌ）、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－アミノビシクロ［１．１．１］ペンタン－１－イル）メチル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート２，２，２－トリフルオロアセテート（ＩＮＸ－Ａ１３－１）（０．３ｇ、０．５０ｍｍｏｌ）、及びＤＭＦ（２ｍＬ）を充填した。この溶液に、ＤＩＰＥＡ（０．１６ｇ、１．２５ｍｍｏｌ）及びＨＡＴＵ（０．２２ｇ、０．６０ｍｍｏｌ）を添加し、室温で１５分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をジエチルエーテルで粉砕し、オフホワイト色の固体として表題化合物を得た（０．４ｇ、７４．９９％）。ＬＣＭＳ：１０６３．７ ［Ｍ＋Ｈ］^＋ (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS , 11aR, 12aS, 12bS) -8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11 ,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)bicyclo[1.1.1 ] Synthesis of tert-butyl pentan-1-yl)amino)-5-oxopentanoate (INX-A13-2)

procedure:
In a 10 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.24 g, 0.50 mmol), 2-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3- aminobicyclo[1.1.1]pentan-1-yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9, 10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate 2,2,2-trifluoroacetate (INX-A13-1) (0.3 g, 0.50 mmol) and DMF (2 mL) were charged. To this solution was added DIPEA (0.16 g, 1.25 mmol) and HATU (0.22 g, 0.60 mmol) and stirred at room temperature for 15 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was triturated with diethyl ether to give the title compound as an off-white solid (0.4 g, 74.99%). LCMS: 1063.7 [M+H] ⁺

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－アセトキシアセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１３－２）（０．４ｇ、０．３７ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ジエチルアミン（０．５５ｇ、７．５ｍｍｏｌ）を添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテル及びペンタンで粉砕して、黄色の固体として表題化合物を得た（０．２２ｇ、粗製）。これを分析することなく次のステップに使用した。 (S)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl -4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[ 1,2-c]pyrrol-10(2H)-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-4-(2-aminoacetamido)-5-oxopentanoic acid tert- Synthesis of butyl (INX-A13-3)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-( (6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7 ,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl ) tert-butyl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5-oxopentanoate (INX-A13-2) (0.4 g, 0.37 mmol) and THF (5 mL). Filled. To this solution was added diethylamine (0.55 g, 7.5 mmol) and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether and pentane to give the title compound as a yellow solid (0.22 g, crude). This was used in the next step without analysis.

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－アセトキシアセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－４－（２－アミノアセトアミド）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１３－３）（０．２２ｇ、０．２６ｍｍｏｌ）及びＤＣＭ（４ｍＬ）を充填した。この溶液に、水（１ｍＬ）中のＮａＨＣＯ_３（０．０６ｇ、０．７８ｍｍｏｌ）溶液及びブロモアセチルブロミド（０．０４ｇ、０．２０ｍｍｏｌ）を滴加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（０．２４ｇ、粗製）。これを精製することなく次のステップに取り込んだ。ＬＣＭＳ：９６３．５ ［Ｍ＋Ｈ］^＋。 (S)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl -4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[ 1,2-c]pyrrol-10(2H)-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-4-(2-(2-bromoacetamido)acetamide)-5- Synthesis of tert-butyl oxopentanoate (INX-A13-4)

procedure:
In a 10 mL single-neck round bottom flask, add (S)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-8b-(2-acetoxyacetyl) -7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-4-(2-amino tert-Butyl acetamido)-5-oxopentanoate (INX-A13-3) (0.22 g, 0.26 mmol) and DCM (4 mL) were charged. To this solution was added dropwise a solution of NaHCO ₃ (0.06 g, 0.78 mmol) in water (1 mL) and bromoacetyl bromide (0.04 g, 0.20 mmol) and stirred at room temperature for 1 h. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as an off-white solid (0.24 g, crude). This was taken into the next step without purification. LCMS: 963.5 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－アセトキシアセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１３－４）（０．１２ｇ、０．１２ｍｍｏｌ）及びメタノール：Ｈ_２Ｏ（９：１、５ｍＬ）を充填した。この溶液に、ＮａＨＣＯ_３（０．０２０ｇ、０．２４ｍｍｏｌ）を添加し、反応混合物を室温で６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライト床を通して濾過した。組み合わせた有機層を真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（０．０８ｇ、７２．４６％）。ＬＣＭＳ：９２０．５ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydro naphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5- Synthesis of tert-butyl oxopentanoate (INX-A13-5)

procedure:
In a 10 mL single-neck round bottom flask, add (S)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-8b-(2-acetoxyacetyl) -7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-4-(2-( tert-Butyl 2-bromoacetamido)acetamido)-5-oxopentanoate (INX-A13-4) (0.12 g, 0.12 mmol) and methanol:H ₂ O (9:1, 5 mL) were charged. To this solution was added NaHCO ₃ (0.020 g, 0.24 mmol) and the reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through a bed of Celite. The combined organic layers were evaporated under vacuum to give the title compound as an off-white solid (0.08g, 72.46%). LCMS: 920.5 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１３－５）（０．０８ｇ、０．０８ｍｍｏｌ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＴＦＡ（０．４ｍｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ、１９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．１％ＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝５８：４２、保持時間１３分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．００７ｇ、１０．１２％）。ＬＣＭＳ：８６３．４［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４８（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．９５－６．８０（ｍ， ３Ｈ）， ６．６０ （ｄ， Ｊ＝８．８ Ｈｚ， １Ｈ）， ６．２６（ｄ， １Ｈ）， ５．９９（ｍ， １Ｈ）， ４．５１－３．５０（ｍ， ８Ｈ）， ３．５１－２．６０（ｍ， ４Ｈ）， ２．５０－２．００（ｓ， ８Ｈ）， ２．００－１．２０（ｍ， ２２Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydro naphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)bicyclo[1.1.1]pentan-1-yl)amino)-5- Synthesis of oxopentanoic acid (INX-A13)

procedure:
In a 10 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR , 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a ,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)bicyclo[1.1.1]pentane tert-Butyl-1-yl)amino)-5-oxopentanoate (INX-A13-5) (0.08 g, 0.08 mmol) and DCM (5 mL) were charged. To this solution was added TFA (0.4 ml) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude material was purified by preparative HPLC (SUNFIRE Prep C18 OBD, 19 x 250 mm, 5 μm, mobile phase: A = 0.1% FA in water, B = acetonitrile, A:B = 58:42, retention time 13 min) to give the title compound as an off-white solid (0.007g, 10.12%). LCMS: 863.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ 7.48 (d, J=10 Hz, 1H), 6.95-6.80 (m, 3H), 6.60 (d, J=8.8 Hz, 1H), 6.26 (d, 1H), 5.99 (m, 1H), 4.51-3.50 (m, 8H), 3.51-2. 60 (m, 4H), 2.50-2.00 (s, 8H), 2.00-1.20 (m, 22H).

（６－（４－ブロモベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３７－１）の合成

手順：
１００ｍｌの一つ口丸底フラスコに、（Ｅ）－（６－（（２－トシルヒドラゾノ）メチル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３２－３）（２．０ｇ、０．４９ｍｍｏｌ）及びジオキサン（８０ｍＬ）を充填した。この溶液に、（４－ブロモフェニル）ボロン酸（１．４７ｇ、０．７３ｍｍｏｌ）及びＫ_２ＣＯ_３（１．０ｇ、０．７３ｍｍｏｌ）を室温で添加し、１００℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル：ヘキサン、５：９５）によって精製し、白色の固体として表題化合物を得た（０．４ｇ、２１．４３％）。ＬＣＭＳ：３８０ ［Ｍ＋Ｈ］^＋。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)methyl)phenyl)-7-hydroxy-8b- (2-Hydroxyacetyl)-6a,8a-dimethyl-1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1 Synthesis of ':4,5]indeno[1,2-c]pyrrol-4(2H)-one (INX-SM-37)

Synthesis of tert-butyl (6-(4-bromobenzyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-37-1)

procedure:
In a 100 ml single-neck round bottom flask, add tert-butyl (E)-(6-((2-tosylhydrazono)methyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-32-3). ) (2.0 g, 0.49 mmol) and dioxane (80 mL). To this solution was added (4-bromophenyl)boronic acid (1.47 g, 0.73 mmol) and K ₂ CO ₃ (1.0 g, 0.73 mmol) at room temperature and stirred at 100° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated NaHCO ₃ solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate:hexane, 5:95) to give the title compound as a white solid (0.4 g, 21.43%). LCMS: 380 [M+H] ⁺ .

手順：
３５ｍＬのガラスバイアルに、（６－（４－ブロモベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３７－１）（０．２ｇ、０．５ｍｍｏｌ）及びジオキサン（６ｍＬ）を充填した。この溶液に、ビス（ピナコラート）ジボロン（０．３９ｇ、０．１５ｍｍｏｌ）及びＫＯＡｃ（０．１ｇ、０．１０ｍｍｏｌ）を添加し、Ｎ_２で３０分間パージした。この溶液に、Ｐｄ（ｄｐｐｆ）Ｃｌ_２・ＣＨ_２Ｃｌ_２（０．０４ｇ、０．０５ｍｍｏｌ）を添加し、１００℃で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル：ヘキサン、５：９５）によって精製し、白色の固体として表題化合物を得た（０．３ｇ、定量的）。ＬＣＭＳ：３２７．２ ［［Ｍ＋Ｈ－Ｂｏｃ］^＋］。 Step 5: (6-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)spiro[3.3]heptan-2-yl)carbamic acid tert -Butyl (INX-SM-37-2)

procedure:
In a 35 mL glass vial, tert-butyl (6-(4-bromobenzyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-37-1) (0.2 g, 0.5 mmol) and dioxane (6 mL). To this solution was added bis(pinacolato)diboron (0.39 g, 0.15 mmol) and KOAc (0.1 g, 0.10 mmol) and purged with _N2 for 30 min. To this solution was added Pd(dppf)Cl ₂ .CH ₂ Cl ₂ (0.04 g, 0.05 mmol) and stirred at 100° C. for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated NaHCO ₃ solution and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate:hexane, 5:95) to give the title compound as a white solid (0.3 g, quantitative). LCMS: 327.2 [[M+H-Boc] ⁺ ].

手順：
２５ｍｌの一つ口丸底フラスコに、（６－（４－（４，４，５，５－テトラメチル－１，３，２－ジオキサボロラン－２－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３７－２）（ＩＢＩＳ－Ｔ－６６－Ａ２）（０．３ｇ、０．７０ｍｍｏｌ）及びアセトン：水（９：１、３ｍＬ）を充填した。この溶液に、ＮａＩＯ_４（１．２ｇ、５．６ｍｍｏｌ）及びＣＨ_３ＣＯＯＮＨ_４（０．４３ｇ、０．５６ｍｍｏｌ）を室温で添加し、５０℃で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（０．１８ｇ、７４．８７％）。ＬＣＭＳ：２４５．７ ［［Ｍ＋Ｈ－Ｂｏｃ］^＋］。 Synthesis of (4-((6-((tert-butoxycarbonyl)amino)spiro[3.3]heptan-2-yl)methyl)phenyl)boronic acid (INX-SM-37-3)

procedure:
In a 25 ml single neck round bottom flask, add (6-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)spiro[3.3]heptane- 2-yl) tert-butyl) carbamate (INX-SM-37-2) (IBIS-T-66-A2) (0.3 g, 0.70 mmol) and acetone:water (9:1, 3 mL) were charged. . To this solution was added NaIO ₄ (1.2 g, 5.6 mmol) and CH ₃ COONH ₄ (0.43 g, 0.56 mmol) at room temperature and stirred at 50° C. for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as an off-white solid (0.18 g, 74.87%). LCMS: 245.7 [[M+H-Boc] ⁺ ].

手順：
３５ｍＬのガラスバイアルに、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート塩酸塩（ＩＮＸ－ＳＭ－３４－２）（０．１２ｇ、０．２８ｍｍｏｌ）、（４－（（６－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）スピロ［３．３］ヘプタン－２－イル）メチル）フェニル）ボロン酸（ＩＮＸ－ＳＭ－３７－３）（０．２９ｇ、０．８４ｍｍｏｌ）及びアセトニトリル（１２ｍＬ）を充填した。この溶液に、ＫＯＨ（０．１５ｇ、２．８０ｍｍｏｌ）及びＣｕ（ＯＡｃ）_２（０．１５ｇ、０．８４ｍｍｏｌ）を添加し、反応混合物を室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライトを通して濾過した。収集した有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：５０：５０）によって精製し、オフホワイト色の固体として表題化合物を得た（０．０６０ｇ、３１．９１％）。ＬＣＭＳ：７２７．６ ［Ｍ＋Ｈ］^＋。 2-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((6-((tert-butoxycarbonyl)amino)spiro[3.3]heptan-2-yl ) methyl) phenyl) -7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradeca Synthesis of hydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-37-4)

procedure:
In a 35 mL glass vial, 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7 ,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl )-2-oxoethyl acetate hydrochloride (INX-SM-34-2) (0.12 g, 0.28 mmol), (4-((6-((tert-butoxycarbonyl)amino)spiro[3.3] Heptan-2-yl)methyl)phenyl)boronic acid (INX-SM-37-3) (0.29 g, 0.84 mmol) and acetonitrile (12 mL) were charged. To this solution, KOH (0.15 g, 2.80 mmol) and Cu(OAc) ₂ (0.15 g, 0.84 mmol) were added and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through Celite. The collected organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 50:50) to give the title compound as an off-white solid (0.060 g, 31.91%). LCMS: 727.6 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）スピロ［３．３］ヘプタン－２－イル）メチル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－３７－４）（０．０６ｇ、０．０８２ｍｍｏｌ）及びメタノール（１ｍＬ）を充填した。この溶液に、ＮａＨＣＯ_３（０．０１３ｇ、０．１６ｍｍｏｌ）を添加し、室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を濃縮して、オフホワイト色の固体として表題化合物を得た（０．０６０ｇ、定量的）。ＬＣＭＳ：５８５．４ ［［Ｍ＋Ｈ］^＋－Ｂｏｃ］。 (6-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1, 4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrole Synthesis of tert-butyl-10(2H)-yl)benzyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-37-5)

procedure:
In a 10 mL single-necked round bottom flask, add 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-((6-((tert-butoxycarbonyl)amino)spiro [3.3] heptan-2-yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11, 11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-SM -37-4) (0.06 g, 0.082 mmol) and methanol (1 mL) were charged. To this solution was added NaHCO ₃ (0.013 g, 0.16 mmol) and stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated to give the title compound as an off-white solid (0.060 g, quantitative). LCMS: 585.4 [[M+H] ⁺ -Boc].

手順：
１０ｍＬの一つ口丸底フラスコに、（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３７－５）（０．０５０ｇ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．５ｍｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．１％ＦＡ、Ｂ＝アセトニトリル、保持時間１０．２７分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．０１１ｇ、２３．４２％）。ＬＣＭＳ：５８５．４ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ） δ：７．４８（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．９５－６．８３（ｍ， ３Ｈ）， ６．５８（ｄ， Ｊ＝８．８ Ｈｚ， １Ｈ）， ６．２６（ｄ， １０．０ Ｈｚ， １Ｈ）， ５．９８（ｓ， １Ｈ）， ４．５０－３．００ （ｍ， ８Ｈ）， ２．６０－１．５５（ｍ，２１Ｈ）， １．５０（ｓ， ３Ｈ）， １．２０－１．０５（ｍ， ２Ｈ）， １．０６（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)methyl)phenyl)-7-hydroxy-8b- (2-Hydroxyacetyl)-6a,8a-dimethyl-1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1 Synthesis of ':4,5]indeno[1,2-c]pyrrol-4(2H)-one (INX-SM-37)

procedure:
In a 10 mL single-neck round bottom flask, add (6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5 ] tert-butyl indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-37-5) (0.050g ) and DCM (2 mL). To this solution was added TFA (0.5 ml) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.1% FA in water, B = acetonitrile, retention time 10.27 min. ) to give the title compound as an off-white solid (0.011 g, 23.42%). LCMS: 585.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD) δ: 7.48 (d, J=10 Hz, 1H), 6.95-6.83 (m, 3H), 6.58 (d, J=8.8 Hz, 1H), 6.26 (d, 10.0 Hz, 1H), 5.98 (s, 1H), 4.50-3.00 (m, 8H), 2 .60-1.55 (m, 21H), 1.50 (s, 3H), 1.20-1.05 (m, 2H), 1.06 (s, 3H).

２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）メチル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－Ａ１０－１）の合成

手順：
２５ｍＬの一つ口丸底フラスコに、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）スピロ［３．３］ヘプタン－２－イル）メチル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－３７－４）（０．３ｇ）及びＤＣＭ（５ｍＬ）を充填した。この溶液に、ＴＦＡ（１ｍＬ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、粗製物をジエチルエーテルによる粉砕によって精製し、オフホワイト色の固体として表題化合物を得た（０．３０ｇ、定量的）。ＬＣＭＳ：６２７．４１［Ｍ＋Ｈ］^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydro naphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxopentane Synthesis of acid (INX-A10)

2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)methyl)phenyl)-7-hydroxy -6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1': Synthesis of 4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-A10-1)

procedure:
In a 25 mL single neck round bottom flask, add 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-((6-((tert-butoxycarbonyl)amino)spiro [3.3] heptan-2-yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11, 11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-SM -37-4) (0.3 g) and DCM (5 mL) were charged. To this solution was added TFA (1 mL) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and the crude was purified by trituration with diethyl ether to give the title compound as an off-white solid (0.30 g, quantitative). LCMS: 627.41 [M+H] ⁺

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．２３０ｇ、０．４７ｍｍｏｌ）、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）メチル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－Ａ１０－１）（０．３ｇ、０．４７ｍｍｏｌ）及びＤＭＦ（６ｍＬ）を充填した。この溶液に、ＤＩＰＥＡ（０．１８５ｇ、１．２５ｍｍｏｌ）及びＨＡＴＵ（０．２７ｇ、０．７１ｍｍｏｌ）を添加し、室温で１５分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水中に注ぎ、固体を濾過し、真空下で乾燥させた。粗製物をシリカゲルカラムクロマトグラフィー（水／アセトニトリル、１３：８７）によって精製し、白色の固体として表題化合物を得た（０．３８０ｇ、７４．０％）。ＬＣＭＳ：１０９１．７ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS , 11aR, 12aS, 12bS) -8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11 ,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzylspiro)[3.3]heptane Synthesis of tert-butyl-2-yl)amino)-5-oxopentanoate (INX-A10-2)

procedure:
In a 25 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.230g, 0.47mmol), 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-((6- Aminospiro[3.3]heptan-2-yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11 , 11a, 12, 12a, 12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX- A10-1) (0.3 g, 0.47 mmol) and DMF (6 mL) were charged. To this solution was added DIPEA (0.185 g, 1.25 mmol) and HATU (0.27 g, 0.71 mmol) and stirred at room temperature for 15 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the solid was filtered and dried under vacuum. The crude material was purified by silica gel column chromatography (water/acetonitrile, 13:87) to give the title compound as a white solid (0.380 g, 74.0%). LCMS: 1091.7 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－アセトキシアセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジルスピロ）［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１０－２）（０．３ｇ、０．２７ｍｍｏｌ）及びＴＨＦ（５４ｍＬ）を充填した。この溶液に、ジエチルアミン（０．２０ｇ、２．７ｍｍｏｌ）を室温で添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテル－ペンタンで粉砕して、白色の固体として表題化合物を得た（０．２２ｇ、９３．７５％）。ＬＣＭＳ：８６９．７ ［Ｍ＋Ｈ］^＋。 (S)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl -4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[ tert-butyl (1,2-c]pyrrol-10(2H)-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-4-(2-aminoacetamido)-5-oxopentanoate ( Synthesis of INX-A10-3)

procedure:
In a 25 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((6-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7 ,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl ) benzylspiro) [3.3] heptan-2-yl) amino) tert-butyl-5-oxopentanoate (INX-A10-2) (0.3 g, 0.27 mmol) and THF (54 mL) were charged. . Diethylamine (0.20 g, 2.7 mmol) was added to this solution at room temperature and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether-pentane to give the title compound as a white solid (0.22 g, 93.75%). LCMS: 869.7 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－アセトキシアセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－４－（２－アミノアセトアミド）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１０－３）（０．２２ｇ、０．２５ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、水（１ｍＬ）に溶解したＮａＨＣＯ_３（０．０５３ｇ、０．５０ｍｍｏｌ）及びブロモアセチルブロミド（０．０７６ｇ、０．３７ｍｍｏｌ）を室温で添加し、３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（０．２１ｇ、８４．８％）。ＬＣＭＳ：９８９．６ ［Ｍ＋Ｈ］^＋。 (S)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl -4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[ 1,2-c]pyrrol-10(2H)-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-4-(2-(2-bromoacetamido)aceto)-5-oxopentane Synthesis of tert-butyl acid (INX-A10-4)

procedure:
In a 25 mL single-neck round bottom flask, add (S)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-8b-(2-acetoxyacetyl) -7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-4-(2-aminoacetamide) tert-Butyl-5-oxopentanoate (INX-A10-3) (0.22 g, 0.25 mmol) and DCM (3 mL) were charged. To this solution was added NaHCO ₃ (0.053 g, 0.50 mmol) and bromoacetyl bromide (0.076 g, 0.37 mmol) dissolved in water (1 mL) at room temperature and stirred for 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as an off-white solid (0.21 g, 84.8%). LCMS: 989.6 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－アセトキシアセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－４－（２－（２－ブロモアセトアミド）アセト）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１０－４）（０．２１０ｇ、０．２１ｍｍｏｌ）及びメタノール（２ｍＬ）を充填した。この溶液に、ＮａＨＣＯ_３（０．０３５ｇ、０．４２ｍｍｏｌ）を添加し、反応混合物を室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライト床を通して濾過した。組み合わせた有機層を濃縮して、オフホワイト色の固体として表題化合物を得た（０．２２０ｇ、定量的）。ＬＣＭＳ：９４７．３ ［Ｍ＋Ｈ］^＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydro naphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxopentane Synthesis of tert-butyl acid (INX-A10-5)

procedure:
In a 10 mL single-neck round bottom flask, add (S)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-8b-(2-acetoxyacetyl) -7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-4-(2-(2- Tert-butyl bromoacetamido)aceto)-5-oxopentanoate (INX-A10-4) (0.210 g, 0.21 mmol) and methanol (2 mL) were charged. To this solution was added NaHCO ₃ (0.035 g, 0.42 mmol) and the reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through a bed of Celite. The combined organic layers were concentrated to give the title compound as an off-white solid (0.220 g, quantitative). LCMS: 947.3 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチルＩＮＸ－Ａ１０－５）（０．２２０ｇ、０．２３２ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、ＴＦＡ（１．５ｍｌ）を添加し、反応混合物を室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（ＳＵＮＦＩＲＥ Ｐｒｅｐ Ｃ１８ ＯＢＤ（２５０×１９）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝５５：４５、保持時間：１５．６７分）によって精製し、白色の固体として表題化合物を得た（０．０２６ｇ、１２．５６％）。ＬＣＭＳ：８９１．５ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ： δ：７．４８（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．９５（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．６２（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．２６（ｄｄ， Ｊ＝ １０．０＆２．０ Ｈｚ， １Ｈ）， ５．９９（ｓ， １Ｈ）， ４．５０－４．２７（ｍ， ４Ｈ）， ４．２０－３．８０ （ｍ， ５Ｈ）， ３．６０－２．９０（ｍ， ４Ｈ）， ２．５０－１．５５（ｍ， ２５Ｈ）， １．５０（ｓ， ３Ｈ）， １．２０－１．００（ｍ， ２Ｈ），１．０７（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((6-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydro naphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxopentane Synthesis of acid (INX-A10)

procedure:
In a 10 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR , 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a ,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)spiro[3.3]heptane-2 tert-Butyl-amino)-5-oxopentanoate INX-A10-5) (0.220 g, 0.232 mmol) and DCM (3 mL) were charged. To this solution was added TFA (1.5 ml) and the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude material was subjected to preparative HPLC (SUNFIRE Prep C18 OBD (250 x 19) mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 55:45, retention time: 15. 67 min) to give the title compound as a white solid (0.026 g, 12.56%). LCMS: 891.5 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD: δ: 7.48 (d, J = 10 Hz, 1H), 6.95 (d, J = 8.4 Hz, 2H), 6 .62(d, J=8.4Hz, 2H), 6.26(dd, J=10.0&2.0Hz, 1H), 5.99(s, 1H), 4.50-4.27(m , 4H), 4.20-3.80 (m, 5H), 3.60-2.90 (m, 4H), 2.50-1.55 (m, 25H), 1.50 (s, 3H) ), 1.20-1.00 (m, 2H), 1.07 (s, 3H).

スキーム２

スキーム３

２－（（６Ｓ，８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ）－６，９－ジフルオロ－１１－ヒドロキシ－１０，１３－ジメチル－３－オキソ－６，７，８，９，１０，１１，１２，１３，１４，１５－デカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－１７－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－２８－１）の合成

手順：
１００ｍＬの密封管に、ジフルプレドナート（５．０ｇ、９．８４ｍｍｏｌ）及びＤＭＦ（５０ｍＬ）を充填した。この溶液に、酢酸カリウム（７．７１ｇ、７．８７ｍｍｏｌ）を添加し、１００℃で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、固体を濾過し、白色の固体として表題化合物を取得した（４．０ｇ、９６．７６％）。ＬＣ／ＭＳ：４２１．３ ［Ｍ＋Ｈ］^＋。 (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxy acetyl)-6a,8a-dimethyl-1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4, 5] Synthesis scheme 1 of indeno[1,2-c]pyrrol-4(2H)-one (INX-SM-28)

Scheme 2

Scheme 3

2-((6S,8S,9R,10S,11S,13S,14S)-6,9-difluoro-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11 , 12,13,14,15-decahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl acetate (INX-SM-28-1)

procedure:
A 100 mL sealed tube was charged with difluprednate (5.0 g, 9.84 mmol) and DMF (50 mL). To this solution was added potassium acetate (7.71 g, 7.87 mmol) and stirred at 100° C. for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the solid was filtered to obtain the title compound as a white solid (4.0 g, 96.76%). LC/MS: 421.3 [M+H] ⁺ .

手順：
１００ｍＬのガラス密封管に、２－（（６Ｓ，８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ）－６，９－ジフルオロ－１１－ヒドロキシ－１０，１３－ジメチル－３－オキソ－６，７，８，９，１０，１１，１２，１３，１４，１５－デカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－１７－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－２８－１）（４．０ｇ、９．５２３ｍｍｏｌ）及び１，４－ジオキサン（４０ｍＬ）を充填した。この反応混合物に、ＴＦＡ（０．０７６ｇ、０．６６６ｍｍｏｌ）及びＮ－ベンジル－１－メトキシ－Ｎ－（（トリメチルシリル）メチル）メタンアミン（２２．５ｇ、９５．２３ｍｍｏｌ）を反応混合物に添加し、１１０℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で濃縮した。粗製物をシリカゲルカラムクロマトグラフィー（ＥｔＯＡｃ／ヘキサン、３２：６８）によって精製し、白色の固体として表題化合物を取得した（３．５ｇ、６６．４５％）。ＬＣ／ＭＳ：５５４．３ ［Ｍ＋Ｈ］^＋。 2-((2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-10-benzyl-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4 , 6a, 6b, 7, 8, 8a, 9, 10, 11, 11a, 12, 12a, 12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrole- Synthesis of 8b(2H)-yl)-2-oxoethyl acetate (INX-SM-28-2)

procedure:
In a 100 mL glass sealed tube, add 2-((6S,8S,9R,10S,11S,13S,14S)-6,9-difluoro-11-hydroxy-10,13-dimethyl-3-oxo-6,7, 8,9,10,11,12,13,14,15-decahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl acetate (INX-SM-28-1) (4.0 g, 9.523 mmol) and 1,4-dioxane (40 mL). To this reaction mixture, TFA (0.076 g, 0.666 mmol) and N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine (22.5 g, 95.23 mmol) were added to the reaction mixture and 110 Stirred at ℃ for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and concentrated under vacuum. The crude was purified by silica gel column chromatography (EtOAc/hexanes, 32:68) to obtain the title compound as a white solid (3.5 g, 66.45%). LC/MS: 554.3 [M+H] ⁺ .

手順：
１００ｍＬのガラス密封管に、２－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－ベンジル－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－２８－２）（３．５ｇ、６．３２９ｍｍｏｌ）及びアセトニトリル（３５ｍＬ）を充填した。この溶液に、ＮａＨＣＯ_３（１．０６ｇ、１２．６６ｍｍｏｌ）及びクロロギ酸１－クロロエチル（１．８１ｇ、１２．６５８ｍｍｏｌ）を室温で添加し、反応混合物を５０℃で４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＥｔＯＡｃで希釈し、Ｈ_２Ｏで洗浄した。有機層をＮａ_２ＳＯ_４によって乾燥させ、減圧下で濃縮した。粗製物を逆相カラムクロマトグラフィー（アセトニトリル：水、２５：７５）によって精製し、白色の固体として表題化合物を取得した（１．６ｇ、５４．６０％）。ＬＣ／ＭＳ：４６４．３ ［Ｍ＋Ｈ］^＋。 2-((2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b ,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrole-8b (2H) Synthesis of -yl)-2-oxoethyl acetate (INX-SM-28-3)

procedure:
In a 100 mL glass sealed tube, 2-((2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-10-benzyl-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl- 4-Oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1 ,2-c]pyrrole-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-28-2) (3.5 g, 6.329 mmol) and acetonitrile (35 mL). To this solution, NaHCO ₃ (1.06 g, 12.66 mmol) and 1-chloroethyl chloroformate (1.81 g, 12.658 mmol) were added at room temperature and the reaction mixture was stirred at 50° C. for 4 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with EtOAc and washed with _H2O . The organic layer was dried with Na ₂ SO ₄ and concentrated under reduced pressure. The crude material was purified by reverse phase column chromatography (acetonitrile:water, 25:75) to obtain the title compound as a white solid (1.6g, 54.60%). LC/MS: 464.3 [M+H] ⁺ .

手順：
２５０ｍＬの一つ口丸底フラスコに、３－ニトロベンゾイルクロリド（１０．０ｇ、０．０５４ｍｏｌ）及びブロモベンゼン（７０ｍＬ）を充填した。この溶液に、塩化アルミニウム（７．１ｇ、０．０５４ｍｍｏｌ）を０℃で添加し、８０℃で２時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物をＨ_２Ｏで希釈し、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、１０：９０）によって精製し、オフホワイト色の固体として表題化合物を得た。（１４．０ｇ、８４．６４％）。^１Ｈ ＮＭＲ（ＤＭＳＯ－ｄ６） δ ８．５１ （ｄ， Ｊ＝８．４Ｈｚ， １Ｈ）， ８．４３（ｓ， １Ｈ）， ８．１６（ｄ， Ｊ＝ ７．６Ｈｚ， １Ｈ）， ７．８７－７．７１（ｍ， ５Ｈ）。 Synthesis of (4-bromophenyl)(3-nitrophenyl)methanone (INX-SM-28-4)

procedure:
A 250 mL single neck round bottom flask was charged with 3-nitrobenzoyl chloride (10.0 g, 0.054 mol) and bromobenzene (70 mL). Aluminum chloride (7.1 g, 0.054 mmol) was added to this solution at 0°C and stirred at 80°C for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with _H2O and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane, 10:90) to give the title compound as an off-white solid. (14.0g, 84.64%). ¹ H NMR (DMSO-d6) δ 8.51 (d, J=8.4Hz, 1H), 8.43 (s, 1H), 8.16 (d, J= 7.6Hz, 1H), 7. 87-7.71 (m, 5H).

手順：
１００ｍＬの一つ口丸底フラスコに、（４－ブロモフェニル）（３－ニトロフェニル）メタノン（ＩＮＸ－ＳＭ－２８－４）（１３．０ｇ、０．０４２ｍｏｌ）及びＴＨＦ（１００ｍＬ）を充填した。この溶液に、水素化ホウ素ナトリウム（１．５ｇ、０．０４２ｍｍｏｌ）を室温で添加し、更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、ゴム状の固体として表題化合物を得た（１３．０ｇ、９９．３５％）。^１Ｈ ＮＭＲ（ＤＭＳＯ－ｄ６） δ ８．２４ （ｓ， １Ｈ）， ８．１０（ｄ， Ｊ＝ ８．４Ｈｚ， １Ｈ）， ７．８１（ｄ， Ｊ＝ ７．６Ｈｚ， １Ｈ）， ７．６０ （ｄｄ， Ｊ＝ ８Ｈｚ， １Ｈ）， ７．５３（ｄ，Ｊ＝８．４Ｈｚ， ２Ｈ）， ７．３８（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．３５（ｂｒｓ， １Ｈ）， ５．８８（ｓ， １Ｈ）。 Synthesis of (4-bromophenyl)(3-nitrophenyl)methanol (INX-SM-28-5)

procedure:
A 100 mL single neck round bottom flask was charged with (4-bromophenyl)(3-nitrophenyl)methanone (INX-SM-28-4) (13.0 g, 0.042 mol) and THF (100 mL). To this solution was added sodium borohydride (1.5 g, 0.042 mmol) at room temperature and stirred for an additional 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a gummy solid (13.0 g, 99.35%). ^1H NMR (DMSO-d6) δ 8.24 (s, 1H), 8.10 (d, J= 8.4Hz, 1H), 7.81 (d, J= 7.6Hz, 1H), 7. 60 (dd, J=8Hz, 1H), 7.53 (d, J=8.4Hz, 2H), 7.38 (d, J=8.4Hz, 2H), 6.35 (brs, 1H), 5.88 (s, 1H).

手順：
２５０ｍＬの一つ口丸底フラスコに、（４－ブロモフェニル）（３－ニトロフェニル）メタノール（ＩＮＸ－ＳＭ－２８－５）（１４．０ｇ、０．０４５ｍｏｌ）及びクロロホルム（５０ｍＬ）を充填した。この溶液に、トリフル酸（２７．０ｇ、０．１８ｍｏｌ）及びトリエチルシラン（５．２７ｇ、０．０４５ｍｏｌ）を０℃で添加し、０℃で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＮａＨＣＯ_３溶液でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：１０：９０）によって精製し、オフホワイト色の固体として表題化合物を得た（８．０ｇ、６０．８％）。^１Ｈ ＮＭＲ（ＤＭＳＯ－ｄ６） δ ８．１１ （ｓ， １Ｈ）， ８．０８（ｄ， Ｊ＝ ８．０Ｈｚ， １Ｈ）， ７．７２（ｄ， Ｊ＝ ７．６Ｈｚ， １Ｈ）， ７．５９ （ｄｄ， Ｊ＝ ８Ｈｚ， １Ｈ）， ７．５１（ｄ，Ｊ＝８．４Ｈｚ， ２Ｈ）， ７．２７（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ４．０８（ｓ， ２Ｈ）。 Synthesis of 1-(4-bromobenzyl)-3-nitrobenzene (INX-SM-28-6)

procedure:
A 250 mL single neck round bottom flask was charged with (4-bromophenyl)(3-nitrophenyl)methanol (INX-SM-28-5) (14.0 g, 0.045 mol) and chloroform (50 mL). Triflic acid (27.0 g, 0.18 mol) and triethylsilane (5.27 g, 0.045 mol) were added to this solution at 0°C, and the mixture was stirred at 0°C for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with NaHCO ₃ solution and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 10:90) to give the title compound as an off-white solid (8.0 g, 60.8%). ^1H NMR (DMSO-d6) δ 8.11 (s, 1H), 8.08 (d, J= 8.0Hz, 1H), 7.72 (d, J= 7.6Hz, 1H), 7. 59 (dd, J=8Hz, 1H), 7.51 (d, J=8.4Hz, 2H), 7.27 (d, J=8.0Hz, 2H), 4.08 (s, 2H).

手順：
２５０ｍＬの一つ口丸底フラスコに、１－（４－ブロモベンジル）－３－ニトロベンゼン（８．０ｇ、０．０２７ｍｏｌ）及びエタノール：水（１：１、１００ｍＬ）を充填し、この溶液に、Ｚｎ粉末（１４．０ｇ、０．２１ｍｍｏｌ）及び（ＩＮＸ－ＳＭ－２８－６）塩化アンモニウム（１１．８ｇ、０．２１ｍｍｏｌ）を室温で添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をセライトを通して濾過し、濾液を真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（７．０ｇ、９８．８％）。ＬＣＭＳ：２６２．１ ［Ｍ＋Ｈ］^＋。 Synthesis of 3-(4-bromobenzyl)aniline (INX-SM-28-7)

procedure:
A 250 mL single neck round bottom flask was charged with 1-(4-bromobenzyl)-3-nitrobenzene (8.0 g, 0.027 mol) and ethanol:water (1:1, 100 mL), and this solution was Zn powder (14.0 g, 0.21 mmol) and (INX-SM-28-6) ammonium chloride (11.8 g, 0.21 mmol) were added at room temperature and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was filtered through Celite and the filtrate was evaporated under vacuum to give the title compound as an off-white solid (7.0 g, 98.8%). LCMS: 262.1 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、３－（４－ブロモベンジル）アニリン（ＩＮＸ－ＳＭ－２８－７）（７．０ｇ、０．０２６ｍｏｌ）及びＴＨＦ（２０ｍＬ）を充填した。この溶液に、ＴＥＡ（５．３ｇ、０．０５３ｍｏｌ）及びｂｏｃ無水物（８．７ｇ、０．０４０ｍｏｌ）を０℃で添加し、室温で１６時間撹拌させた。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：１０：９０）によって精製し、オフホワイト色の固体として表題化合物を得た（１４ｇ、粗製）。これを更に精製することなく次のステップに取り込んだ。ＬＣＭＳ：３６１．７［［Ｍ＋Ｈフラグメンテーション］。 Synthesis of tert-butyl (3-(4-bromobenzyl)phenyl)carbamate (INX-SM-28-8)

procedure:
A 10 mL single neck round bottom flask was charged with 3-(4-bromobenzyl)aniline (INX-SM-28-7) (7.0 g, 0.026 mol) and THF (20 mL). To this solution was added TEA (5.3 g, 0.053 mol) and boc anhydride (8.7 g, 0.040 mol) at 0° C. and allowed to stir at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 10:90) to give the title compound as an off-white solid (14 g, crude). This was taken into the next step without further purification. LCMS: 361.7 [[M+H fragmentation].

手順：
２５ｍＬの一つ口丸底フラスコに、窒素下で（３－（４－ブロモベンジル）フェニル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－２８－８）（１４．０ｇ、０．０３８ｍｏｌ）及び１，４－ジオキサン（３０ｍＬ）を充填した。この溶液に、ビスピナコロンジボラン（ｂｉｓｐｉｎａｃｏｌｏｎｅｄｉｂｏｒａｎｅ）（１４．４ｇ、０．０５７ｍｏｌ）及び酢酸カリウム（７．４ｇ、０．０７６ｍｏｌ）を添加し、Ｎ_２で１５分間パージした。ＰｄＣｌ_２（ｄｐｐｆ）．ＤＣＭ（３．１ｇ、０．００３８ｍｏｌ）を添加し、１１０℃で２時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を室温で冷却し、酢酸エチルで希釈し、セライト床を通して濾過した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：２０：８０）によって精製し、オフホワイト色の半固体として表題化合物を得た（２０．０ｇ、粗製）。ＬＣＭＳ：４１０．４ ［Ｍ＋Ｈ］^＋。 Synthesis of tert-butyl (3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)phenyl)carbamate (INX-SM-28-9)

procedure:
In a 25 mL single neck round bottom flask under nitrogen, tert-butyl (3-(4-bromobenzyl)phenyl)carbamate (INX-SM-28-8) (14.0 g, 0.038 mol) and 1, Charged with 4-dioxane (30 mL). To this solution was added bispinacolonediborane (14.4 g, 0.057 mol) and potassium acetate (7.4 g, 0.076 mol) and purged with _N2 for 15 minutes. _PdCl2 (dppf). DCM (3.1 g, 0.0038 mol) was added and heated at 110° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was cooled to room temperature, diluted with ethyl acetate, and filtered through a bed of Celite. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 20:80) to give the title compound as an off-white semi-solid (20.0 g, crude). LCMS: 410.4 [M+H] ⁺ .

手順：
１０００ｍＬの一つ口丸底フラスコに、（３－（４－（４，４，５，５－テトラメチル－１，３，２－ジオキサボロラン－２－イル）ベンジル）フェニル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－２８－９）（２０．０ｇ、０．０４８ｍｏｌ）及びアセトン：水（９：１、２００ｍＬ）を充填した。この溶液に、ＮａＩＯ_４（８１．７ｇ、０．３８ｍｏｌ）及び酢酸アンモニウム（２９．２６ｇ、０．３８ｍｏｌ）を添加し、還流で２時間加熱した。ＴＬＣによって示される反応の完了後、反応混合物を室温まで冷却し、酢酸エチルで希釈し、セライトを通して濾過した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をｎ－ペンテン及びジエチルエーテルで粉砕し、オフホワイト色の固体として表題化合物を得た（１５．０ｇ、９５．５％）。ＬＣＭＳ：３２８．２ ［［Ｍ＋Ｈ］^＋ Synthesis of (4-(3-((tert-butoxycarbonyl)amino)benzyl)phenyl)boronic acid (INX-SM-28-10)

procedure:
In a 1000 mL single-neck round bottom flask, add tert-butyl (3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)phenyl)carbamate ( INX-SM-28-9) (20.0 g, 0.048 mol) and acetone:water (9:1, 200 mL) were charged. To this solution was added NaIO ₄ (81.7 g, 0.38 mol) and ammonium acetate (29.26 g, 0.38 mol) and heated at reflux for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was cooled to room temperature, diluted with ethyl acetate, and filtered through Celite. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude was triturated with n-pentene and diethyl ether to give the title compound as an off-white solid (15.0 g, 95.5%). LCMS: 328.2 [[M+H] ⁺

手順：
３５ｍＬのガラスバイアルに、２－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－２８－３）（０．３２０ｇ、０．６９１ｍｍｏｌ）及びアセトニトリル（３５ｍＬ）を充填した。この溶液に、（４－（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ベンジル）フェニル）ボロン酸（ＩＮＸ－ＳＭ－２８－１０）（１．０１ｇ、３．１０９ｍｍｏｌ）、ＫＯＨ（０．３８７ｇ、６．９１０ｍｍｏｌ）及びＣｕ（ＯＡＣ）_２（０．３７５ｇ、２．０７３ｍｍｏｌ）を添加し、６０℃で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＥｔＯＡｃで希釈し、セライトを通して濾過し、真空下で濃縮した。粗製物をシリカカラムクロマトグラフィー（酢酸エチル／ヘキサン、３０：７０）によって精製し、黄色の固体として表題化合物を得た（０．０９５ｇ、１８．５９％）を得た。
ＬＣ／ＭＳ：７４５．４ ［Ｍ＋Ｈ］^＋。 2-((2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-(3-((tert-butoxycarbonyl)amino)benzyl)phenyl)-2,6b-difluoro -7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2' Synthesis of ,1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-28-11)

procedure:
In a 35 mL glass vial, 2-((2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1 ,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c] Pyrrole-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-28-3) (0.320 g, 0.691 mmol) and acetonitrile (35 mL) were charged. To this solution, (4-(3-((tert-butoxycarbonyl)amino)benzyl)phenyl)boronic acid (INX-SM-28-10) (1.01 g, 3.109 mmol), KOH (0.387 g, 6.910 mmol) and Cu(OAC) ₂ (0.375 g, 2.073 mmol) were added and stirred at 60° C. for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with EtOAc, filtered through Celite, and concentrated under vacuum. The crude material was purified by silica column chromatography (ethyl acetate/hexanes, 30:70) to give the title compound as a yellow solid (0.095 g, 18.59%).
LC/MS: 745.4 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、２－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ベンジル）フェニル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－２８－１１）（０．０９５ｇ、０．１２７ｍｍｏｌ）及びメタノール（２ｍＬ）を充填した。この溶液に、炭酸カリウム（０．０２７ｇ、０．１９１ｍｍｏｌ）を添加し、反応混合物を室温で３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＥｔＯＡｃで希釈し、Ｈ_２Ｏで洗浄した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で濃縮して、黄色の固体として粗製の表題化合物生成物を得た（０．０８５ｇ、９５．２％）。ＬＣＭＳ：７０３．４ ［Ｍ＋Ｈ］^＋ (3-(4-((2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a- Dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno Synthesis of tert-butyl [1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)carbamate (INX-SM-28-12)

procedure:
In a 10 mL one-necked round bottom flask, add 2-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-(3-((tert-butoxycarbonyl)amino)). benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a , 12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-28-11) (0.095 g, 0.127 mmol) and methanol (2 mL). To this solution was added potassium carbonate (0.027 g, 0.191 mmol) and the reaction mixture was stirred at room temperature for 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with EtOAc and washed with _H2O . The combined organic layers were dried over Na ₂ SO ₄ and concentrated under vacuum to give the crude title compound product as a yellow solid (0.085 g, 95.2%). LCMS:703.4 [M+H] ⁺

手順：
１０ｍＬの一つ口丸底フラスコに、（３－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）フェニル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－２８－１２）（０．０８５ｇ、０．１２１ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．１ｍＬ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で濃縮した。粗製物を分取ＨＰＬＣ（カラム：Ｃ１８、２５０×２１．２ｍｍ、５μｍ、移動相Ａ＝水中の０．０５％トリフルオロ酢酸、Ｂ＝アセトニトリル：メタノール：ＩＰＡ（６５：２５：１０）、Ａ：Ｂ＝６５：３５、保持時間１５．３２分）によって精製し、黄色の固体として表題化合物を取得した（０．００５ｇ、６．８６％）。ＬＣ／ＭＳ：６０３．３ ［Ｍ＋Ｈ］^＋ (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxy acetyl)-6a,8a-dimethyl-1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4, 5] Synthesis of indeno[1,2-c]pyrrol-4(2H)-one (INX-SM-28)

procedure:
In a 10 mL single neck round bottom flask, add (3-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2 tert-butyl',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)carbamate (INX-SM-28-12) (0.085g, 0 .121 mmol) and DCM (2 mL). To this solution was added TFA (0.1 mL) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated under vacuum. The crude material was subjected to preparative HPLC (column: C18, 250 x 21.2 mm, 5 μm, mobile phase A = 0.05% trifluoroacetic acid in water, B = acetonitrile:methanol:IPA (65:25:10), A: B=65:35, retention time 15.32 minutes) to obtain the title compound as a yellow solid (0.005 g, 6.86%). LC/MS: 603.3 [M+H] ⁺

２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（４－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ベンジル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－４０－１）の合成

手順：
３５ｍＬのガラスバイアルに、アセトリル（４０ｍＬ）中の２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート塩酸塩（ＩＮＸ－ＳＭ－３４－２）（０．４ｇ、０．９３ｍｍｏｌ）及び（４－（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ベンジル）フェニル）ボロン酸（ＩＮＸ－ＳＭ－２８－１０）（１．０７ｇ、３．２７ｍｍｏｌ）を充填した。この溶液に、ＫＯＨ（０．５２ｇ、９．３６ｍｍｏｌ）及びＣｕ（ＯＡｃ）_２（０．５０ｇ、２．８１ｍｍｏｌ）を添加し、反応混合物を室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライトを通して濾過した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル：ヘキサン、３０：７０）によって精製し、オフホワイト色の固体として表題化合物を得た（０．１ｇ、１５．８％）。ＬＣＭＳ：７０９．５ ［Ｍ＋Ｈ］^＋。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-(3-aminobenzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl -1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2- c] Synthesis of pyrrol-4(2H)-one (INX-SM-40)

2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-(4-((tert-butoxycarbonyl)amino)benzyl)phenyl)-7-hydroxy-6a,8a -dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5] Synthesis of indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-40-1)

procedure:
In a 35 mL glass vial, 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4) in acetyl (40 mL) was added. , 6a, 6b, 7, 8, 8a, 9, 10, 11, 11a, 12, 12a, 12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrole- 8b(2H)-yl)-2-oxoethyl acetate hydrochloride (INX-SM-34-2) (0.4 g, 0.93 mmol) and (4-(3-((tert-butoxycarbonyl)amino)benzyl ) Phenyl)boronic acid (INX-SM-28-10) (1.07 g, 3.27 mmol) was charged. To this solution, KOH (0.52 g, 9.36 mmol) and Cu(OAc) ₂ (0.50 g, 2.81 mmol) were added and the reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through Celite. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate:hexane, 30:70) to give the title compound as an off-white solid (0.1 g, 15.8%). LCMS: 709.5 [M+H] ⁺ .

手順：
２５ｍＬの一つ口丸底フラスコに、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（４－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ベンジル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテー（ＩＮＸ－ＳＭ－４０－１）（０．１３ｇ、０．１８ｍｍｏｌ）及びメタノール：水（２ｍＬ）を充填した。この溶液に、ＮａＨＣＯ_３（０．０３０ｇ、０．３６ｍｍｏｌ）を添加し、反応混合物を室温で１２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライトを通して濾過した。組み合わせた有機層を蒸発させて、オフホワイト色の固体として粗ＩＮＸ－ＳＭ－４０－２生成物を得た（０．０６ｇ、４９．９８％）。ＭＳ：６６７．４ ［Ｍ＋Ｈ］^＋。 (4-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1, 4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrole Synthesis of tert-butyl-10(2H)-yl)benzyl)phenyl)carbamate (INX-SM-40-2)

procedure:
In a 25 mL single-neck round bottom flask, add 2-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-(4-((tert-butoxycarbonyl)amino)benzyl) phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[ 2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-40-1) (0.13 g, 0. 18 mmol) and methanol:water (2 mL). To this solution was added NaHCO ₃ (0.030 g, 0.36 mmol) and the reaction mixture was stirred at room temperature for 12 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through Celite. The combined organic layers were evaporated to give the crude INX-SM-40-2 product as an off-white solid (0.06 g, 49.98%). MS: 667.4 [M+H] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（４－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）フェニル）カルバミン酸ｔｅｒｔ－ブチルＩＮＸ－ＳＭ－４０－２）（０．０６ｇ、０．０８ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．１２ｍＬ）を添加し、反応混合物を室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝６６：３４、保持時間１４．５分）によって精製し、オフホワイト色の固体として表題化合物を得た（０．００４ｇ、８．８２％）。ＬＣＭＳ：５６７ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ： δ：７．４８（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ７．３９（ｔ， Ｊ＝８．０Ｈｚ， １Ｈ）， ７．２５（ｄ， Ｊ＝７．６Ｈｚ， １Ｈ）， ７．１１（ｄ， Ｊ＝７．６Ｈｚ １Ｈ）， ７．０６（ｓ， １Ｈ）， ７．０３（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．６２（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．２６（ｄｄ， Ｊ＝ １０．０ ＆ ２．０Ｈｚ， １Ｈ）， ５．９９（ｓ， １Ｈ）， ４．６０－４．３０（ｍ， ３Ｈ）， ３．９１（ｓ， ２Ｈ）， ３．６０－３．００（ｍ， ４Ｈ）， ２．７０－１．５５（ｍ， １０Ｈ）， １．５０（ｓ， ３Ｈ）， １．１０（ｓ， ３Ｈ）， １．１０－０．９５（ｍ， ２Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-(3-aminobenzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl -1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2- c] Synthesis of pyrrol-4(2H)-one (INX-SM-40)

procedure:
In a 10 mL single-neck round bottom flask, add (4-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5 tert-butylindeno[1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)carbamate INX-SM-40-2) (0.06 g, 0.08 mmol) and DCM (2 mL). Filled. To this solution was added TFA (0.12 mL) and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 66: 34, retention time 14.5 minutes) to give the title compound as an off-white solid (0.004 g, 8.82%). LCMS: 567 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD: δ: 7.48 (d, J = 10 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.25 (d, J=7.6Hz, 1H), 7.11 (d, J=7.6Hz 1H), 7.06 (s, 1H), 7.03 (d, J=8.4Hz, 2H), 6.62 (d, J=8.4Hz, 2H), 6.26 (dd, J=10.0 & 2.0Hz, 1H), 5.99 (s, 1H), 4.60-4.30 (m, 3H), 3.91 (s, 2H), 3.60-3.00 (m, 4H), 2.70-1.55 (m, 10H), 1.50 (s, 3H), 1.10 (s, 3H), 1.10-0.95 (m, 2H).

（Ｅ）－６－（（２－トシルヒドラゾノ）メチル）－２－アザスピロ［３．３］ヘプタン－２－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４７－１）の合成

手順：
３５ｍＬのガラスバイアルに、窒素雰囲気下で６－ホルミル－２－アザスピロ［３．３］ヘプタン－２－カルボン酸ｔｅｒｔ－ブチル（０．２ｇ、０．８８０ｍｍｏｌ）及びＥｔＯＨ（１０ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホンヒドラジド（０．２４７ｇ、１．３３ｍｍｏｌ）及び触媒ＡｃＯＨ（０．１ｍＬ）を添加し、室温で０．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、固体沈殿物を濾過し、真空下で乾燥させて、白色の固体として表題化合物を得た（０．３ｇ、８５．８８％）。ＬＣＭＳ：３９４．２ ［Ｍ＋Ｈ］＋。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((2-Azaspiro[3.3]heptan-6-yl)methyl)phenyl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-47)

(E) Synthesis of tert-butyl-6-((2-tosylhydrazono)methyl)-2-azaspiro[3.3]heptane-2-carboxylate (INX-SM-47-1)

procedure:
A 35 mL glass vial was charged with tert-butyl 6-formyl-2-azaspiro[3.3]heptane-2-carboxylate (0.2 g, 0.880 mmol) and EtOH (10 mL) under nitrogen atmosphere. To this solution was added p-toluenesulfone hydrazide (0.247 g, 1.33 mmol) and catalytic AcOH (0.1 mL) and stirred at room temperature for 0.5 h. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the solid precipitate was filtered and dried under vacuum to give the title compound as a white solid (0.3 g, 85.88%). LCMS: 394.2 [M+H]+.

手順：
３５ｍＬのガラスバイアルに、窒素雰囲気下で（Ｅ）－６－（（２－トシルヒドラゾノ）メチル）－２－アザスピロ［３．３］ヘプタン－２－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４７－１）（０．３ｇ、０．７６２ｍｍｏｌ）及びジオキサン（５ｍＬ）を充填した。この溶液に、（４－ホルミルフェニル）ボロン酸（０．１７１ｇ、１．１４ｍｍｏｌ）及びＫ２ＣＯ３（０．１５８ｇ、１．１４ｍｍｏｌ）を室温で添加し、１００℃で更に３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させた。粗製物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：５０：５０）によって精製し、黄色のゴム状固体として表題化合物を得た（０．０６５ｇ、２７．８６％）。ＬＣＭＳ：３１６．２ ［Ｍ＋Ｈ］＋。 Synthesis of tert-butyl 6-(4-formylbenzyl)-2-azaspiro[3.3]heptane-2-carboxylate (INX-SM-47-2)

procedure:
In a 35 mL glass vial, tert-butyl (E)-6-((2-tosylhydrazono)methyl)-2-azaspiro[3.3]heptane-2-carboxylate (INX-SM-47-1) was added under nitrogen atmosphere. ) (0.3 g, 0.762 mmol) and dioxane (5 mL). To this solution was added (4-formylphenyl)boronic acid (0.171 g, 1.14 mmol) and K2CO3 (0.158 g, 1.14 mmol) at room temperature and stirred for an additional 3 hours at 100°C. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under vacuum. The crude material was purified by silica gel column chromatography (ethyl acetate/hexane: 50:50) to give the title compound as a yellow gummy solid (0.065 g, 27.86%). LCMS: 316.2 [M+H]+.

手順：
２５ｍＬの一つ口丸底フラスコに、６－（４－ホルミルベンジル）－２－アザスピロ［３．３］ヘプタン－２－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４７－２）（０．０６５ｇ、０．２０６ｍｍｏｌ）、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］］フェナレン－３－オン（１６－α－ヒドロキシプレドニゾロン）（０．０７７ｇ、０．２０６ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＭｇＳＯ４（０．１０３ｇ、１．０３ｍｍｏｌ）及びＨＣｌＯ４（０．１２３ｇ、１．０３ｍｍｏｌ）を添加し、室温で更に１．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ３溶液でクエンチし、真空下で濃縮した。粗製物を冷水で粉砕し、参加したものを濾過し、真空下で乾燥させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－ＰＡＣＫ ＯＤＳ－ＡＱ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル：メタノール：２－プロパノール（６５：２５：１０）、Ａ：Ｂ＝５８：４２、保持時間１２．２３分）によって精製し、白色の固体として表題化合物を得た（０．０２５ｇ、１７．７７％）。ＬＣＭＳ：５７４．５ ［Ｍ＋Ｈ］＋；１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）：δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３７（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．１８（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０＆ ２．０ Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４６（ｓ， アセタールＨ， １Ｈ）， ５．０７（ｄ， Ｊ＝５．２Ｈｚ，Ｃ１６－Ｈ １Ｈ）， ４．６３ （ｄ，Ｊ＝１９．６Ｈｚ， １Ｈ）， ４．４５－４．４４（ｓ， １Ｈ）， ４．３３（ｄ， Ｊ＝１９．６Ｈｚ １Ｈ）， ４．０７（ｓ， ２Ｈ）， ３．９５（ｓ， ２Ｈ）， ２．７５－１．７３（ｍ， １６Ｈ）， １．５０（ｓ， ３Ｈ）， １．０５－１．０５（ｍ， ２Ｈ）， １．０１（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((2-Azaspiro[3.3]heptan-6-yl)methyl)phenyl)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1' Synthesis of :4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-47)

procedure:
In a 25 mL single-neck round bottom flask, add tert-butyl 6-(4-formylbenzyl)-2-azaspiro[3.3]heptane-2-carboxylate (INX-SM-47-2) (0.065 g, 0.206 mmol), (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7 , 8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]]phenalen-3-one (16-α-hydroxyprednisolone) (0.077 g, 0. 206 mmol) and DCM (2 mL). To this solution were added MgSO4 (0.103 g, 1.03 mmol) and HClO4 (0.123 g, 1.03 mmol) and stirred for an additional 1.5 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated NaHCO3 solution and concentrated under vacuum. The crude was triturated with cold water and the residue was filtered and dried under vacuum. The crude product was subjected to preparative HPLC (column: YMC-PACK ODS-AQ Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile: methanol: 2- Purification by propanol (65:25:10), A:B=58:42, retention time 12.23 min) gave the title compound as a white solid (0.025 g, 17.77%). LCMS: 574.5 [M+H]+; 1H NMR (400 MHz, MeOD): δ: 7.47 (d, J = 10.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H) , 7.18 (d, J=8.4Hz, 2H), 6.27 (dd, J=10.0&2.0Hz, 1H), 6.04 (s, 1H), 5.46 (s, Acetal H, 1H), 5.07 (d, J=5.2Hz, C16-H 1H), 4.63 (d, J=19.6Hz, 1H), 4.45-4.44 (s, 1H) ), 4.33 (d, J=19.6Hz 1H), 4.07 (s, 2H), 3.95 (s, 2H), 2.75-1.73 (m, 16H), 1.50 (s, 3H), 1.05-1.05 (m, 2H), 1.01 (s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－アザスピロ［３．３］ヘプタン－２－イル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１９－１）の合成

手順：
５０ｍＬの一つ口丸底フラスコに、ＤＭＦ（６ｍＬ）中の（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．６０５ｇ、１．２５ｍｍｏｌ）及びＨＡＴＵ（０．５９６ｇ、１．５６ｍｍｏｌ）を充填した。この溶液に、ＤＩＰＥＡ（０．４０５ｇ、３．１３ｍｍｏｌ）、続いて、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（２－アザスピロ［３．３］ヘプタン－６－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－４７）（０．６ｇ、１．０４ｍｍｏｌ）を室温で添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗製物を逆相カラムクロマトグラフィーによって精製し（ＡＣＮ：水、７０：３０）、白色の固体として表題化合物を得た（０．２０ｇ、１８．４２％）。ＬＣＭＳ：１０３８．８ ［Ｍ＋Ｈ］＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-(6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-azaspiro[3.3]heptan-2-yl)-5-oxo Synthesis of pentanoic acid (INX-A19)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a, 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-azaspiro[3. 3] Synthesis of tert-butyl heptane-2-yl)-5-oxopentanoate (INX-A19-1)

procedure:
In a 50 mL single neck round bottom flask, add (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert) in DMF (6 mL). -Butoxy)-5-oxopentanoic acid (INX-P-4) (0.605 g, 1.25 mmol) and HATU (0.596 g, 1.56 mmol) were charged. To this solution was added DIPEA (0.405 g, 3.13 mmol) followed by (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((2-Azaspiro[3 .3] heptan-6-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a , 12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-47) (0 .6 g, 1.04 mmol) was added at room temperature and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under vacuum to obtain the crude product. The crude material was purified by reverse phase column chromatography (ACN:water, 70:30) to give the title compound as a white solid (0.20g, 18.42%). LCMS: 1038.8 [M+H]+.

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－アザスピロ［３．３］ヘプタン－２－イル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１９－１）（０．２ｇ、０．１９２ｍｍｏｌ）及びＴＨＦ（２ｍＬ）を充填した。この溶液に、ジエチルアミン（０．１４０ｇ、１．９２ｍｍｏｌ）を添加し、室温で２．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で濃縮した。粗製物をＤＣＭ及びヘキサンで粉砕し、白色の固体として表題化合物を得た（０．１２ｇ、７６．３４％）。ＬＣＭＳ：８１６．６ ［Ｍ＋Ｈ］＋。 (S)-4-(2-aminoacetamide)-5-(6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2 -hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1' tert-butyl:4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-azaspiro[3.3]heptan-2-yl)-5-oxopentanoate Synthesis of (INX-A19-2)

procedure:
In a 25 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(6-(4-(( 6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b, 7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl ) Filled with tert-butyl)-2-azaspiro[3.3]heptan-2-yl)-5-oxopentanoate (INX-A19-1) (0.2 g, 0.192 mmol) and THF (2 mL). did. To this solution was added diethylamine (0.140 g, 1.92 mmol) and stirred at room temperature for 2.5 hours. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated under vacuum. The crude was triturated with DCM and hexane to give the title compound as a white solid (0.12g, 76.34%). LCMS: 816.6 [M+H]+.

手順：
２５ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－アミノアセトアミド）－５－（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－アザスピロ［３．３］ヘプタン－２－イル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１９－２）（０．１２０ｇ、０．１４７ｍｍｏｌ）及びＤＣＭ（１ｍＬ）を充填した。この溶液に、水（０．１ｍＬ）に溶解したＮａ２ＣＯ３（０．０３１ｇ、０．２９４ｍｍｏｌ）、続いてブロモアセチルブロミド（０．０２９ｇ、０．１４７ｍｍｏｌ）を室温で添加し、０．５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（０．０９５ｇ、６８．９５％）。ＬＣＭＳ：Ｃ４８Ｈ６３７９ＢｒＦＮ３Ｏ１１に対する計算値（９３６．３６）、測値９３６．４［Ｍ＋Ｈ］＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-(6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-azaspiro[3.3]heptan-2-yl)-5-oxo Synthesis of tert-butyl pentanoate (INX-A19-3)

procedure:
In a 25 mL single neck round bottom flask, (S)-4-(2-aminoacetamide)-5-(6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS) )-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-azaspiro[3.3]heptan-2-yl )-5-oxopentanoate (INX-A19-2) (0.120 g, 0.147 mmol) and DCM (1 mL) were charged. To this solution was added Na2CO3 (0.031 g, 0.294 mmol) dissolved in water (0.1 mL) followed by bromoacetyl bromide (0.029 g, 0.147 mmol) at room temperature and stirred for 0.5 h. . After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na2SO4 and evaporated under vacuum to give the title compound as an off-white solid (0.095g, 68.95%). LCMS: Calcd for C48H6379BrFN3O11 (936.36), found 936.4 [M+H]+.

手順：
２５ｍＬの一つ口丸底フラスコに、ＤＣＭ（３ｍＬ）中の（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）－２－アザスピロ［３．３］ヘプタン－２－イル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ１９－３）（０．０９５ｇ、０．１０１ｍｍｏｌ）を充填した。この溶液に、ＴＦＡ（０．２ｍｌ）を室温で添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＮＥＷ Ｘ－ｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ（２５０×１９）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝ＡＣＮ、Ａ：Ｂ＝６８：３２、保持時間１３．５９分）により精製して、白色の固体として表題化合物を得た（０．００６ｇ、６．７２％）。ＬＣＭＳ：Ｃ４４Ｈ５５７９ＢｒＦＮ３Ｏ１１に対する計算値（８８０．３）、実測値８８０．２［Ｍ＋Ｈ］＋；１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ ７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３６（ｄ， Ｊ＝７．６Ｈｚ， ２Ｈ）， ７．２０－７．１７（ｍ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０ ＆ ２．０Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４５（ｓ， １Ｈ）， ５．０６（ｄ， Ｊ＝５．２Ｈｚ， １Ｈ）， ５．００－３．８０（ｍ， １２Ｈ）， ２．７５－１．６５（ｍ， ２０Ｈ）， １．５１（ｓ， ３Ｈ）， １．１８－１．０５（ｍ， ２Ｈ）， １．０３ （ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-(6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-azaspiro[3.3]heptan-2-yl)-5-oxo Synthesis of pentanoic acid (INX-A19)

procedure:
In a 25 mL single neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide)-5-(6-(4-((6aR,6bS,7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a, 8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2- Tert-butyl azaspiro[3.3]heptan-2-yl)-5-oxopentanoate (INX-A19-3) (0.095 g, 0.101 mmol) was charged. TFA (0.2 ml) was added to this solution at room temperature and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: NEW 32, retention time 13.59 min) to give the title compound as a white solid (0.006 g, 6.72%). LCMS: Calculated value for C44H5579BrFN3O11 (880.3), actual value 880.2 [M+H]+; 1H NMR (400 MHz, MeOD): δ 7.47 (d, J=10.0Hz, 1H), 7.36 (d, J=7.6Hz, 2H), 7.20-7.17(m, 2H), 6.27(dd, J=10.0 & 2.0Hz, 1H), 6.04(s, 1H), 5.45 (s, 1H), 5.06 (d, J=5.2Hz, 1H), 5.00-3.80 (m, 12H), 2.75-1.65 (m, 20H), 1.51 (s, 3H), 1.18-1.05 (m, 2H), 1.03 (s, 3H).

（（１ｒ，４ｒ）－４－（ヒドロキシメチル）シクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１４－１）の合成

手順：
１００ｍＬの一つ口丸底フラスコ中で、（１ｒ，４ｒ）－４－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）シクロヘキサン－１－カルボン酸（３．０ｇ、１２．３４ｍｍｏｌ）をＴＨＦ（３０ｍＬ）中に溶解し、ボランジメチルスルフィド（ＢＨ３．ＤＭＳ）（３．０ｍＬ、６．１７ｍｍｏｌ）を０℃で添加した。反応混合物を０℃で３０分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を希釈物でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をブラインで洗浄し、Ｎａ２ＳＯ４上で乾燥させ、真空下で蒸発させて、白色の固体として表題化合物を得た（３．０ｇ、粗製）。それを精製せずに次のステップに使用した。ＬＣＭＳ：２３０．２ ［Ｍ＋Ｈ］＋。 (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(((1r,4R)-4-aminocyclohexyl)methyl)phenyl)-2,6b-difluoro -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[ Synthesis of 2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-14)

Synthesis of tert-butyl ((1r,4r)-4-(hydroxymethyl)cyclohexyl)carbamate (INX-SM-14-1)

procedure:
In a 100 mL single neck round bottom flask, (1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexane-1-carboxylic acid (3.0 g, 12.34 mmol) was dissolved in THF (30 mL). Dissolve and add borane dimethyl sulfide (BH3.DMS) (3.0 mL, 6.17 mmol) at 0°C. The reaction mixture was stirred at 0°C for 30 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with diluent and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4 and evaporated under vacuum to give the title compound as a white solid (3.0 g, crude). It was used in the next step without purification. LCMS: 230.2 [M+H]+.

手順：
１００ｍＬの一つ口丸底フラスコ中に、ＤＣＭ（３０ｍＬ）中の（（１ｒ，４ｒ）－４－（ヒドロキシメチル）シクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１４－１）（３．０ｇ、１３．０８ｍｍｏｌ）の溶液を添加した。この溶液に、ＤＭＰ（６．６ｇ、１５．７２ｍｍｏｌ）を０℃で添加し、室温で更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＮａＨＣＯ３溶液でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をブラインで洗浄し、Ｎａ２ＳＯ４上で乾燥させ、真空下で蒸発させて、白色の固体として粗製の表題化合物を得た（３．０ｇ、粗製）。それを分析せずに次のステップに直接使用した。 Synthesis of tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate (INX-SM-14-2)

procedure:
In a 100 mL single neck round bottom flask, add tert-butyl ((1r,4r)-4-(hydroxymethyl)cyclohexyl)carbamate (INX-SM-14-1) (3.0 g) in DCM (30 mL). , 13.08 mmol) was added. DMP (6.6 g, 15.72 mmol) was added to this solution at 0° C., and the mixture was further stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with NaHCO3 solution and extracted with DCM. The combined organic layers were washed with brine, dried over Na2SO4 and evaporated under vacuum to give the crude title compound as a white solid (3.0 g, crude). It was used directly in the next step without analysis.

手順：
１００ｍＬの一つ口丸底フラスコに、（（１ｒ，４ｒ）－４－ホルミルシクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１４－２）（３．０ｇ、１３．２１ｍｍｏｌ）及びエタノール（３０ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホンヒドラジド（３．６ｇ、１９．８２ｍｍｏｌ）及び触媒量のＡｃＯＨ（０．１ｍＬ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、形成された固体を濾過し、真空下で乾燥させて、白色の固体として表題化合物を得た（３．５ｇ、６７．０５％）。ＬＣＭＳ：３９６．２ ［Ｍ＋Ｈ］＋。 Synthesis of tert-butyl ((1r,4r)-4-((E)-(2-tosylhydrazono)methyl)cyclohexyl)carbamate (INX-SM-14-3)

procedure:
In a 100 mL single-neck round bottom flask, tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate (INX-SM-14-2) (3.0 g, 13.21 mmol) and ethanol (30 mL) were added. filled with. To this solution was added p-toluenesulfone hydrazide (3.6 g, 19.82 mmol) and a catalytic amount of AcOH (0.1 mL) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the solid formed was filtered and dried under vacuum to give the title compound as a white solid (3.5 g, 67.05%) . LCMS: 396.2 [M+H]+.

手順：
１００ｍＬの一つ口丸底フラスコに、（（１ｒ，４ｒ）－４－（（Ｅ）－（２－トシルヒドラゾノ）メチル）シクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１４－３）（２．０ｇ、５．０６ｍｍｏｌ）及びジオキサン（２０ｍＬ）を充填した。この溶液に、Ｋ２ＣＯ３（１．０ｇ、７．５０ｍｍｏｌ）を添加し、反応混合物をＮ２で２０分間パージした。この溶液に、（４－ホルミルフェニル）ボロン酸（１．１３ｇ、７．５８ｍｍｏｌ）を添加し、更に２時間還流した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させた。粗生成物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン：５０：５０）によって精製して、黄色の粘着性固体として表題化合物を得た（０．６ｇ、３７．３８％）。ＬＣＭＳ：３１８．３ ［Ｍ＋Ｈ］＋。 Synthesis of tert-butyl ((1r,4r)-4-(4-formylbenzyl)cyclohexyl)carbamate (INX-SM-14-4)

procedure:
In a 100 mL single-neck round bottom flask, add tert-butyl ((1r,4r)-4-((E)-(2-tosylhydrazono)methyl)cyclohexyl)carbamate (INX-SM-14-3) (2. 0 g, 5.06 mmol) and dioxane (20 mL). To this solution was added K2CO3 (1.0 g, 7.50 mmol) and the reaction mixture was purged with N2 for 20 minutes. To this solution was added (4-formylphenyl)boronic acid (1.13 g, 7.58 mmol) and refluxed for an additional 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under vacuum. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane: 50:50) to give the title compound as a yellow sticky solid (0.6 g, 37.38%). LCMS: 318.3 [M+H]+.

手順：
１０ｍｌのガラスバイアルに、ＤＣＭ（２ｍＬ）中の（（１ｒ，４ｒ）－４－（４－ホルミルベンジル）シクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１４－４）（０．１０ｇ、０．３１ｍｍｏｌ）及び（６Ｓ，８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－６，９－ジフルオロ－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（ＩＮＸ－ＳＭ－２５－１）（０．１３ｇ、０．３１ｍｍｏｌ）を充填した。この溶液に、ＭｇＳＯ４（０．１８ｇ、１．５０ｍｍｏｌ）及びＨＣｌＯ４（０．２６ｇ、２．６ｍｍｏｌ）を０℃で添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ３溶液でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０ Ｘ ２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝７０：３０、保持時間１５．９２分）により精製して、白色の固体として表題化合物を得た（０．０１５ｇ、７．７８％）。ＬＣＭＳ：６１２．３ ［Ｍ＋Ｈ］＋；１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ－ｄ６）： δ７．６２ （ｂｒｓ， ３Ｈ）， ７．３５（ｄ， Ｊ＝８Ｈｚ， ２Ｈ）， ７．２７（ｄ， Ｊ＝９．６Ｈｚ， １Ｈ）， ７．１８（ｄ， Ｊ＝８Ｈｚ， ２Ｈ）， ６．３０（ｄｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．１３（ｓ， １Ｈ）， ５．７０－５．６０（ｍ， １Ｈ）， ５．５６（ｂｒ ｓ， １Ｈ）， ５． ５．４６（ｓ， アセタール－Ｈ， １Ｈ）， ５．１４（ｔ， １Ｈ）， ５．０９（ｄ， Ｊ＝４．４Ｈｚ， Ｃ１６－Ｈ， １Ｈ）， ４．５３－４．４８（ｍ， １Ｈ）， ４，２４－４．１８（ｍ， ２Ｈ）， ２．９４－２．８８（ｍ， １Ｈ）， ２．６０－１．４０（ｍ， １５Ｈ）， １．４５（ｓ， ３Ｈ）， １．２３－０．９８（ｍ， ４Ｈ）， ０．８７（ｓ， ３Ｈ）。 (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(((1r,4R)-4-aminocyclohexyl)methyl)phenyl)-2,6b-difluoro -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[ Synthesis of 2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-14)

procedure:
In a 10 ml glass vial was added tert-butyl ((1r,4r)-4-(4-formylbenzyl)cyclohexyl)carbamate (INX-SM-14-4) (0.10 g, 0.0 g in DCM (2 mL)). 31 mmol) and (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13 -dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (INX-SM-25-1) ( 0.13 g, 0.31 mmol). To this solution was added MgSO4 (0.18 g, 1.50 mmol) and HClO4 (0.26 g, 2.6 mmol) at 0 C and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated NaHCO3 solution and extracted with DCM. The combined organic layers were dried over Na2SO4 and evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 X 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 70: 30, retention time 15.92 minutes) to give the title compound as a white solid (0.015 g, 7.78%). LCMS: 612.3 [M+H]+; 1H NMR (400 MHz, DMSO-d6): δ7.62 (brs, 3H), 7.35 (d, J=8Hz, 2H), 7.27 (d, J =9.6Hz, 1H), 7.18 (d, J=8Hz, 2H), 6.30 (dd, J=10Hz, 1H), 6.13 (s, 1H), 5.70-5.60 (m, 1H), 5.56 (br s, 1H), 5. 5.46 (s, acetal-H, 1H), 5.14 (t, 1H), 5.09 (d, J=4.4Hz, C16-H, 1H), 4.53-4.48 (m , 1H), 4,24-4.18 (m, 2H), 2.94-2.88 (m, 1H), 2.60-1.40 (m, 15H), 1.45 (s, 3H) ), 1.23-0.98 (m, 4H), 0.87 (s, 3H).

（（１ｓ，４ｓ）－４－ヒドロキシメチル）シクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１５－１）の合成
ＩＮＸ－ＳＭ－１５－１
手順：
５０ｍｌの丸底フラスコに、（１ｓ，４ｓ）－４－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）シクロヘキサン－１－カルボン酸（２．５ｇ、１０．２８ｍｍｏｌ）及びＴＨＦ（２５ｍＬ）を充填した。この溶液に、ボラン．ＤＭＳ（ＴＨＦ中２Ｍ）（１２．３４ｍＬ、３０．６０ｍｍｏｌ）を０℃で添加し、室温で更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＭｅＯＨ及び希釈ＨＣｌでクエンチした。反応混合物を酢酸エチルで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させて、白色の固体として表題化合物を得た（２．３ｇ、９７．６１％）。１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ－ｄ６）： δ ６．６７ （ｄ， Ｊ＝ ７．２ Ｈｚ， １Ｈ）， ４．３５ （ｔ， Ｊ＝ ５．６ Ｈｚ， １Ｈ）， ３．４４ （ｂｒ ｓ， １Ｈ）， ３．２５ （ｔ， Ｊ＝ ６．０ Ｈｚ， １Ｈ）， １．５０－１．４３ （ｍ， １９Ｈ）。 (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(((1s,4S)-4-aminocyclohexyl)methyl)phenyl)-2,6b-difluoro -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[ Synthesis scheme of 2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-15)

Synthesis of tert-butyl ((1s,4s)-4-hydroxymethyl)cyclohexyl)carbamate (INX-SM-15-1) INX-SM-15-1
procedure:
A 50 ml round bottom flask was charged with (1s,4s)-4-((tert-butoxycarbonyl)amino)cyclohexane-1-carboxylic acid (2.5 g, 10.28 mmol) and THF (25 mL). Add borane to this solution. DMS (2M in THF) (12.34 mL, 30.60 mmol) was added at 0° C. and stirred for an additional hour at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with MeOH and dilute HCl. The reaction mixture was extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under vacuum to give the title compound as a white solid (2.3g, 97.61%). 1H NMR (400 MHz, DMSO-d6): δ 6.67 (d, J= 7.2 Hz, 1H), 4.35 (t, J= 5.6 Hz, 1H), 3.44 (br s , 1H), 3.25 (t, J = 6.0 Hz, 1H), 1.50-1.43 (m, 19H).

手順：
５０ｍｌの一つ口丸底フラスコに、（（１ｓ，４ｓ）－４－ヒドロキシメチル）シクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１５－１）（２．３ｇ、１０．０３ｍｍｏｌ）及びＤＣＭ（２５ｍＬ）を充填した。この溶液に、ＤＭＰ（６．４ｇ、１５．０９ｍｍｏｌ）を室温で添加し、更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＮａＨＣＯ_３溶液でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をブラインで洗浄し、Ｎａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、淡黄色のゴム状固体として表題化合物を得た（２．３ｇ粗製）。それを分析せずに次のステップで使用した。 Synthesis of tert-butyl ((1s,4s)-4-formylcyclohexyl)carbamate (INX-SM-15-2)

procedure:
In a 50 ml single neck round bottom flask, tert-butyl ((1s,4s)-4-hydroxymethyl)cyclohexyl)carbamate (INX-SM-15-1) (2.3 g, 10.03 mmol) and DCM ( 25 mL). DMP (6.4 g, 15.09 mmol) was added to this solution at room temperature and further stirred for 2 hours. After the completion of the reaction as indicated by TLC, the reaction mixture was quenched with NaHCO ₃ solution and extracted with DCM. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a pale yellow gummy solid (2.3 g crude). It was used in the next step without analysis.

Synthesis of tert-butyl ((1s,4s)-4-((E)-(2-tosylhydrazono)methyl)cyclohexyl)carbamate (INX-SM-15-3) INX-SM-15-3
procedure:
In a 25 mL single-neck round bottom flask, tert-butyl ((1s,4s)-4-formylcyclohexyl)carbamate (INX-SM-15-2) (2.3 g, 10.12 mmol) and EtOH (25 mL) were added. filled with. To this solution, p-toluenesulfone hydrazide (1.88 g, 10.12 mmol) and acetic acid (catalyst) were added and stirred for an additional 2 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane, 1:1) to give the title compound as a white solid (2.5 g, 62.47%). LCMS: 394.4 [MH]+.

手順：
２５ｍＬの一つ口丸底フラスコに、（（１ｓ，４ｓ）－４－（（Ｅ）－（２－トシルヒドラゾノ）メチル）シクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１５－３）（０．８５ｇ、２．１５ｍｍｏｌ）及びジオキサン（１０ｍＬ）を充填した。この溶液に、（４－ホルミルフェニル）ボロン酸（０．３０ｇ、２．０２ｍｍｏｌ）及びＫ_２ＣＯ_３（０．４５ｇ、３．２３ｍｍｏｌ）を添加し、１００℃で更に２時間還流した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、１：１）によって精製して、淡黄色のゴム状固体として表題化合物を得た（０．１５ｇ、２１．９９％）。ＬＣＭＳ：３１８．３ ［Ｍ＋Ｈ］＋。 Synthesis of tert-butyl ((1s,4s)-4-(4-formylbenzyl)cyclohexyl)carbamate (INX-SM-15-4)

procedure:
In a 25 mL single-necked round bottom flask, add tert-butyl ((1s,4s)-4-((E)-(2-tosylhydrazono)methyl)cyclohexyl)carbamate (INX-SM-15-3) (0. 85 g, 2.15 mmol) and dioxane (10 mL). To this solution was added (4-formylphenyl)boronic acid (0.30 g, 2.02 mmol) and K ₂ CO ₃ (0.45 g, 3.23 mmol) and refluxed at 100° C. for an additional 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was purified by silica gel column chromatography (ethyl acetate/hexanes, 1:1) to give the title compound as a pale yellow gummy solid (0.15 g, 21.99%). LCMS: 318.3 [M+H]+.

手順：
１０ｍＬの一つ口丸底フラスコに、（（１ｓ，４ｓ）－４－（４－ホルミルベンジル）シクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１５－４）（０．１５ｇ、０．４７ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、（６Ｓ，８Ｓ，９Ｒ，１０Ｓ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－６，９－ジフルオロ－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（ＩＮＸ－ＳＭ－２５－１）（０．１５ｇ、０．３７ｍｍｏｌ）、ＭｇＳＯ４（０．２８ｇ、２．３６ｍｍｏｌ）、及びＨＣｌＯ４（０．２３ｇ、２．３６ｍｍｏｌ）を添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＮａＨＣＯ３でクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＮＥＷ Ｘ－ｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ（２５０×１９）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝８０：２０、保持時間１０．００分）により精製して、白色の固体として表題化合物を得た（０．０３０ｇ、１０．３８％）。ＬＣＭＳ：６１２．２ ［Ｍ＋Ｈ］＋；１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ－ｄ６）：δ：７．６９（ｂｒ ｓ， ３Ｈ）， ７．３６（ｄ， Ｊ＝１０．０， ２Ｈ）， ７．２８（ｄ， Ｊ＝１０．２， １Ｈ）， ７．１９（ｄ， Ｊ＝１０．０， ２Ｈ）， ６．３０（ｄｄ， Ｊ＝１０．０ ＆ ２．０Ｈｚ， ２Ｈ）， ５．７４－５．６０（ｍ， ２Ｈ）， ５．５６（ｂｒ ｓ， １Ｈ）， ５．４６ （ｓ， アセタール－Ｈ， １Ｈ）， ５．１４（ｔ， Ｊ＝６．０， １Ｈ）， ４．９６（ｄ， Ｊ＝４．４Ｈｚ， Ｃ１６－Ｈ， １Ｈ）， ４．６５－４．２０（ｍ， ３Ｈ）， ３．２５－３．１０（ｍ， １Ｈ）， ２．６２－２．１．６０（ｍ， １２Ｈ）， １．５０（ｓ， ３Ｈ）， １．４６－１．３０（ｍ， ４Ｈ）， １．２０ （ｓ， ３Ｈ）。 (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(((1s,4S)-4-aminocyclohexyl)methyl)phenyl)-2,6b-difluoro -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[ Synthesis of 2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-15)

procedure:
In a 10 mL single-neck round bottom flask, add tert-butyl ((1s,4s)-4-(4-formylbenzyl)cyclohexyl)carbamate (INX-SM-15-4) (0.15 g, 0.47 mmol). and DCM (3 mL). To this solution, (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10, 13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (INX-SM-25-1) (0.15 g, 0.37 mmol), MgSO4 (0.28 g, 2.36 mmol), and HClO4 (0.23 g, 2.36 mmol) were added and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with NaHCO3 and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under vacuum. The crude product was subjected to preparative HPLC (column: NEW 20, retention time 10.00 min) to give the title compound as a white solid (0.030 g, 10.38%). LCMS: 612.2 [M+H]+; 1H NMR (400 MHz, DMSO-d6): δ: 7.69 (br s, 3H), 7.36 (d, J=10.0, 2H), 7. 28 (d, J=10.2, 1H), 7.19 (d, J=10.0, 2H), 6.30 (dd, J=10.0 & 2.0Hz, 2H), 5.74 -5.60 (m, 2H), 5.56 (br s, 1H), 5.46 (s, acetal-H, 1H), 5.14 (t, J=6.0, 1H), 4. 96 (d, J=4.4Hz, C16-H, 1H), 4.65-4.20 (m, 3H), 3.25-3.10 (m, 1H), 2.62-2.1 .60 (m, 12H), 1.50 (s, 3H), 1.46-1.30 (m, 4H), 1.20 (s, 3H).

２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－イル）メチル）ベンジル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテートカルバメート（ＩＮＸ－ＳＭ－１７－１）の合成

手順：
３５ｍＬのガラスバイアルに、ジオキサン（５ｍＬ）中の２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（０．５ｇ、１．１７ｍｍｏｌ）（ＩＮＸ－ＳＭ－３４－２）及び（３－（４－ホルミルベンジル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（０．３５２ｇ、１．１７ｍｍｏｌ）（ＩＮＸ－ＳＭ－３－５）を充填した。この溶液に、ＡｃＯＨ（０．０７０ｇ、１．１６ｍｍｏｌ）及びＮａＣＮＢＨ３（０．０８８ｇ、１．４ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＥｔＯＡＣで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物をシリカゲルカラムクロマトグラフィー（ヘキサン／酢酸エチル、３０：７０）によって精製して、白色の固体として表題化合物を得た（０．１ｇ、１２％）。ＬＣＭＳ：７１３．７ ［Ｍ＋Ｈ］＋。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)benzyl)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2 Synthesis of ',1':4,5]indeno[1,2-c]pyrrol-4(2H)-one (INX-SM-17)

2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-((3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentane-1 -yl)methyl)benzyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b- Synthesis of tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate carbamate (INX-SM-17-1)

procedure:
In a 35 mL glass vial, 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4) in dioxane (5 mL) was added. , 6a, 6b, 7, 8, 8a, 9, 10, 11, 11a, 12, 12a, 12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrole- 8b(2H)-yl)-2-oxoethyl acetate (0.5 g, 1.17 mmol) (INX-SM-34-2) and (3-(4-formylbenzyl)bicyclo[1.1.1]pentane) tert-butyl-1-yl)carbamate (0.352 g, 1.17 mmol) (INX-SM-3-5) was charged. To this solution were added AcOH (0.070 g, 1.16 mmol) and NaCNBH3 (0.088 g, 1.4 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with EtOAC. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was purified by silica gel column chromatography (hexane/ethyl acetate, 30:70) to give the title compound as a white solid (0.1 g, 12%). LCMS: 713.7 [M+H]+.

手順：
１０ｍＬの一つ口丸底フラスコに、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－イル）メチル）ベンジル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテートカルバメート（ＩＮＸ－ＳＭ－１７－１）（０．１ｇ、０．１４ｍｍｏｌ）及びメタノール（２ｍＬ）を充填した。この溶液に、ＮａＨＣＯ_３（０．０２３ｇ、０．２８ｍｍｏｌ）を添加し、室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライトを通して濾過した。組み合わせた有機層を濃縮して、オフホワイト色の固体として粗製の表題化合物の生成物を得た（０．０８０ｇ、８５．０１％）。ＬＣＭＳ：６７１．５ ［Ｍ＋Ｈ］＋。 (3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1 ,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c] Synthesis of tert-butyl pyrrol-10(2H)-yl)methyl)benzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-17-2)

procedure:
In a 10 mL single-neck round bottom flask, add 2-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-((tert-butoxycarbonyl)amino)bicyclo). [1.1.1] Pentan-1-yl)methyl)benzyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10, 11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate carbamate ( INX-SM-17-1) (0.1 g, 0.14 mmol) and methanol (2 mL) were charged. To this solution was added NaHCO ₃ (0.023 g, 0.28 mmol) and stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through Celite. The combined organic layers were concentrated to give the crude title compound product as an off-white solid (0.080 g, 85.01%). LCMS: 671.5 [M+H]+.

手順：
１０ｍＬの一つ口丸底フラスコに、（３－（４－（（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）メチル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１７－２）（０．０８０ｇ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（１ｍＬ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗生成物をジエチルエーテル及びｎ－ペンタンで粉砕して、白色の固体として表題化合物を得た（０．０７０ｇ、定量的）。ＬＣＭＳ：５７１．４ ［Ｍ＋Ｈ］＋；１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．４２（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ７．２５（ｄ， Ｊ＝１０．０Ｈｚ， ２Ｈ）， ６．２８（ｄ， Ｊ＝９．２Ｈｚ， １Ｈ）， ６．０５（ｓ， １Ｈ）， ４．４８－４．２６（ｍ， ５Ｈ）， ３．８７－３．７７（ｍ， ２Ｈ），３．６０－３．２０（ｍ， ２Ｈ）， ２．９６（ｓ， ２Ｈ）， ２．９０－２．６０（ｍ， ２Ｈ）， ２．５０－２．００（ｍ， ４Ｈ）， １．８９（ｓ， ６Ｈ）， １．７０－１．５５（ｍ， ２Ｈ）， １．５１（ｓ， ３Ｈ）， １．３０－１．１９（ｍ， ３Ｈ）， １．０７（ｓ， ３Ｈ） (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)benzyl)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2 Synthesis of ',1':4,5]indeno[1,2-c]pyrrol-4(2H)-one 2,2,2-trifluoroacetate (INX-SM-17)

procedure:
In a 10 mL single-neck round bottom flask, add (3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a , 8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4, 5] tert-butyl indeno[1,2-c]pyrrol-10(2H)-yl)methyl)benzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-17-2 ) (0.080 g) and DCM (2 mL). TFA (1 mL) was added to this solution at room temperature and stirred for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was triturated with diethyl ether and n-pentane to give the title compound as a white solid (0.070 g, quantitative). LCMS: 571.4 [M+H]+; 1H NMR (400 MHz, MeOD): δ: 7.47 (d, J=10.0Hz, 1H), 7.42 (d, J=8.0Hz, 2H) , 7.25 (d, J=10.0Hz, 2H), 6.28 (d, J=9.2Hz, 1H), 6.05 (s, 1H), 4.48-4.26 (m, 5H), 3.87-3.77 (m, 2H), 3.60-3.20 (m, 2H), 2.96 (s, 2H), 2.90-2.60 (m, 2H) , 2.50-2.00 (m, 4H), 1.89 (s, 6H), 1.70-1.55 (m, 2H), 1.51 (s, 3H), 1.30-1 .19 (m, 3H), 1.07 (s, 3H)

２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（３－アミノベンジル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－２９－Ａ－１）の合成

手順：
１０ｍＬの一つ口丸底フラスコに、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（４－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ベンジル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－４０－１）（０．３８ｇ、０．５３ｍｍｏｌ）及びＤＣＭ（１ｍＬ）を充填した。この溶液に、ＴＦＡ（１．２ｍＬ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、粉砕して、オフホワイト色の固体として表題化合物を得た（０．２２ｇ、６７．４２％）。ＬＣＭＳ：６０９．４ ［Ｍ＋Ｈ］＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydro Synthesis of naphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)amino)-5-oxopentanoic acid (INX-A29)

2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1 ,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c] Synthesis of pyrrole-8b(2H)-yl)-2-oxoethyl acetate (INX-29-A-1)

procedure:
In a 10 mL one-neck round bottom flask, add 2-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-(4-((tert-butoxycarbonyl)amino)benzyl). phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[ 2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-40-1) (0.38 g, 0.53 mmol ) and DCM (1 mL). To this solution was added TFA (1.2 mL) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated to give the title compound as an off-white solid (0.22 g, 67.42%). LCMS: 609.4 [M+H]+.

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．３９ｇ、０．８５ｍｍｏｌ）、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（３－アミノベンジル）フェニル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－２９Ａ－１）（０．５ｇ、０．８５ｍｍｏｌ）及びＤＭＦ（２ｍＬ）を充填した。この溶液に、ＤＩＰＥＡ（０．２６ｇ、２．０５ｍｍｏｌ）及びＨＡＴＵ（０．３７ｇ、０．９８ｍｍｏｌ）を添加し、室温で１５分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させた。粗生成物をジエチルエーテルで粉砕して、オフホワイト色の固体として表題化合物を得た（０．２６ｇ、２９．４７％）。ＬＣＭＳ １０７３．９ ［Ｍ＋Ｈ］＋。 (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((4-(4-((6aR,6bS,7S,8aS,8bS , 11aR, 12aS, 12bS) -8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11 ,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)amino)-5- Synthesis of tert-butyl oxopentanoate (INX-29-A-2)

procedure:
In a 10 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.39 g, 0.85 mmol), 2-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-(3-amino benzyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydro naphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-29A-1) (0.5g, 0.85mmol ) and DMF (2 mL). To this solution was added DIPEA (0.26 g, 2.05 mmol) and HATU (0.37 g, 0.98 mmol) and stirred at room temperature for 15 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under vacuum. The crude product was triturated with diethyl ether to give the title compound as an off-white solid (0.26 g, 29.47%). LCMS 1073.9 [M+H]+.

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（４－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－アセトキシアセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）フェニル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－２９－Ａ－２）（０．２６ｇ、０．２４ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ジエチルアミン（０．１７ｇ、２．４ｍｍｏｌ）を添加し、２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、ジエチルエーテル及びペンタンで粉砕して、オフホワイト色の固体として表題化合物を得た（０．１４ｇ、６７．８９％）。ＬＣＭＳ：８５１ ［Ｍ＋Ｈ］＋。 (S)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl -4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[ Synthesis of tert-butyl 1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)amino)-4-(2-aminoacetamido)-5-oxopentanoate (INX-29-A-3)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((4-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7 ,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl ) tert-butyl)benzyl)phenyl)amino)-5-oxopentanoate (INX-29-A-2) (0.26 g, 0.24 mmol) and THF (5 mL) were charged. To this solution was added diethylamine (0.17 g, 2.4 mmol) and stirred for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and triturated with diethyl ether and pentane to give the title compound as an off-white solid (0.14 g, 67.89%). LCMS: 851 [M+H]+.

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－アセトキシアセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）フェニル）アミノ）－４－（２－アミノアセトアミド）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－２９－Ａ－３）（０．１４ｇ、０．１６ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、水（１ｍＬ）中のＮａＨＣＯ_３（０．０４ｇ、０．４９ｍｍｏｌ）溶液及びブロモアセチルブロミド（０．０５ｇ、０．２４ｍｍｏｌ）を室温で滴加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させて、オフホワイト色の固体として表題化合物を得た（０．１８ｇ、定量的）。ＬＣＭＳ：Ｃ５１Ｈ６４７９ＢｒＦＮ４Ｏ１０に対する計算値（９７１．３８）、実測値９７１．８０［Ｍ＋Ｈ］＋。 (S)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-acetoxyacetyl)-7-hydroxy-6a,8a-dimethyl -4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[ tert-butyl 1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)amino)-4-(2-(2-bromoacetamido)acetamido)-5-oxopentanoate (INX-29-A -4) Synthesis

procedure:
In a 10 mL single-neck round bottom flask, add (S)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-8b-(2-acetoxyacetyl) -7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2' , 1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)amino)-4-(2-aminoacetamido)-5-oxopentanoate tert-butyl( INX-29-A-3) (0.14 g, 0.16 mmol) and DCM (2 mL) were charged. To this solution was added a solution of NaHCO ₃ (0.04 g, 0.49 mmol) in water (1 mL) and bromoacetyl bromide (0.05 g, 0.24 mmol) dropwise at room temperature and stirred for 1 h. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na2SO4 and evaporated under vacuum to give the title compound as an off-white solid (0.18 g, quantitative). LCMS: Calcd for C51H6479BrFN4O10 (971.38), found 971.80 [M+H]+.

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－アセトキシアセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）フェニル）アミノ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－２９－Ａ－４）（０．１８ｇ、０．１８ｍｍｏｌ）及びメタノール：Ｈ２Ｏ（９：１、２ｍＬ）を充填した。この溶液に、ＮａＨＣＯ３（０．０３ｇ、０．３７ｍｍｏｌ）を添加し、室温で６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライトベッドを通して濾過した。組み合わせた有機層を濃縮して、オフホワイトの固体として粗製の表題化合物の生成物を得た（０．１７ｇ、９８．７１％）。ＬＣＭＳ：Ｃ４９Ｈ６２７９ＢｒＦＮ４Ｏ９に対する計算値（９２９．３７）、実測値９２９．７０［Ｍ＋Ｈ］＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydro tert-butyl naphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)amino)-5-oxopentanoate (INX-29-A -5) Synthesis

procedure:
In a 10 mL single-neck round bottom flask, add (S)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-8b-(2-acetoxyacetyl) -7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)amino)-4-(2-(2-bromoacetamido)acetamido)-5-oxopentane Tert-butyl acid (INX-29-A-4) (0.18 g, 0.18 mmol) and methanol:H2O (9:1, 2 mL) were charged. To this solution was added NaHCO3 (0.03 g, 0.37 mmol) and stirred at room temperature for 6 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through a bed of Celite. The combined organic layers were concentrated to give the crude title compound product as an off-white solid (0.17 g, 98.71%). LCMS: Calcd for C49H6279BrFN4O9 (929.37), found 929.70 [M+H]+.

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０（２Ｈ）－イル）ベンジル）フェニル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－２９－Ａ－５）（０．１７ｇ、０．１８ｍｍｏｌ）及びＤＣＭ（１ｍＬ）を充填した。この溶液に、ＴＦＡ（０．８５ｍＬ）を室温で添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－５μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝ＣＡＮ、Ａ：Ｂ＝５５：４５、保持時間１４分）により精製して、白色の固体として表題化合物を得た（０．０２５ｇ、１５．６５％）。ＬＣＭＳ：Ｃ４５Ｈ５４７９ＢｒＦＮ４Ｏ９に対する計算値（８７３．３１）、実測値８７３．２０ ［Ｍ＋Ｈ］ ＋；１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）：δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．４２－７．３７（ｍ， ２Ｈ）， ７．２０（ｄ， Ｊ＝８．０Ｈｚ， １Ｈ）， ７．０３（ｄ， Ｊ＝８．４Ｈｚ， ２Ｈ）， ６．９３（ｄ， Ｊ＝７．２Ｈｚ， ＝１Ｈ）， ６．６４（ｄ， Ｊ＝８．４Ｈｚ， ＝２Ｈ）， ６．２６ （ｄｄ， Ｊ＝１０．０ ＆ １．６Ｈｚ， １Ｈ）， ５．９９（ｓ， １Ｈ）， ４．５３－４．２６（ｍ， ４Ｈ）， ３．９３－３．９２（ｍ， ３Ｈ）， ３．８３（ｓ， ２Ｈ）， ３．５７－２．９８（ｍ， ６Ｈ）， ２．７０－１．５６（ｍ， １２Ｈ）， １．５０（ｓ， ３Ｈ）， １．０７（ｍ， ５Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy- 8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a,12,12a,12b-tetradecahydro Synthesis of naphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)amino)-5-oxopentanoic acid (INX-29-A)

procedure:
In a 10 mL single neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,11aR , 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,8b,9,11,11a ,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-10(2H)-yl)benzyl)phenyl)amino)-5-oxopentane Tert-butyl acid (INX-29-A-5) (0.17 g, 0.18 mmol) and DCM (1 mL) were charged. To this solution was added TFA (0.85 mL) at room temperature and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude material was subjected to preparative HPLC (YMC-Actus Triart Prep C18-S, 250 x 20 mm S-5 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = CAN, A:B = 55:45, Purification (retention time 14 min) gave the title compound as a white solid (0.025 g, 15.65%). LCMS: Calculated value for C45H5479BrFN4O9 (873.31), actual value 873.20 [M+H] +; 1H NMR (400 MHz, MeOD): δ: 7.47 (d, J=10.0Hz, 1H), 7. 42-7.37 (m, 2H), 7.20 (d, J=8.0Hz, 1H), 7.03 (d, J=8.4Hz, 2H), 6.93 (d, J=7 .2Hz, =1H), 6.64 (d, J=8.4Hz, =2H), 6.26 (dd, J=10.0 & 1.6Hz, 1H), 5.99 (s, 1H) , 4.53-4.26 (m, 4H), 3.93-3.92 (m, 3H), 3.83 (s, 2H), 3.57-2.98 (m, 6H), 2 .70-1.56 (m, 12H), 1.50 (s, 3H), 1.07 (m, 5H).

４－（（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－イル）メチル）安息香酸メチル（ＩＮＸ－ＳＭ－１６－１）の合成

手順：
１００ｍＬの一つ口丸底フラスコに、（Ｅ）－（３－（（２－トシルヒドラゾノ）メチル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３－４）（１．０ｇ、２．６４ｍｍｏｌ）を充填し、ジオキサン（２０ｍＬ）及びＫ２ＣＯ３（０．５７６ｇ、３．９５ｍｍｏｌ）を、白色のｐｐｔが観察されるまでＮ２（ｇ）で脱気して添加した。４－（メトキシカルボニル）フェニル）ボロン酸（０．７５ｇ、３．９５ｍｍｏｌ）を添加し、１１０℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させて、粗生成物を得た。粗生成物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、１：１）によって精製して、無色の液体として表題化合物を得た（０．４ｇ、４５．８０％）。１Ｈ ＮＭＲ （ＣＤＣｌ３） δ：７．９７ （ｄ， Ｊ＝ ８．４ Ｈｚ， ２Ｈ，）， ７．１８ （ｄ， Ｊ＝８ Ｈｚ， ２Ｈ）， ４．８９ （ｓ， １Ｈ）， ３．９２ （ｓ， ３Ｈ）， ２．８９ （ｓ， ２Ｈ）， １．８３（ｓ， ６Ｈ）， １．４６（ｓ， ９Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)benzoyl)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2 Synthesis scheme of ',1':4,5]indeno[1,2-c]pyrrol-4(2H)-one (INX-SM-16)

Synthesis of methyl 4-((3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentan-1-yl)methyl)benzoate (INX-SM-16-1)

procedure:
In a 100 mL single-neck round bottom flask, add tert-butyl (E)-(3-((2-tosylhydrazono)methyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-3). -4) (1.0 g, 2.64 mmol) and degassed with N2 (g) until white ppt was observed. Added. 4-(methoxycarbonyl)phenyl)boronic acid (0.75 g, 3.95 mmol) was added and stirred at 110° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under vacuum to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane, 1:1) to give the title compound as a colorless liquid (0.4 g, 45.80%). 1H NMR (CDCl3) δ: 7.97 (d, J = 8.4 Hz, 2H,), 7.18 (d, J = 8 Hz, 2H), 4.89 (s, 1H), 3.92 (s, 3H), 2.89 (s, 2H), 1.83 (s, 6H), 1.46 (s, 9H).

手順：
１００ｍＬの一つ口丸底フラスコに、４－（（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－イル）メチル）安息香酸メチル（ＩＮＸ－ＳＭ－１６－１）（０．９ｇ、２．７２ｍｍｏｌ）及びＴＨＦ：Ｈ２Ｏ（１：１、６ｍＬ）を充填した。この溶液に、ＬｉＯＨ・Ｈ２Ｏ（０．６５ｇ、１６．３ｍｍｏｌ）を添加し、５０℃で５時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、１０％ＭｅＯＨ：ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、白色の固体として表題化合物を得た。（０．８ｇ、９２．８２％）。１Ｈ ＮＭＲ （ＤＭＳＯ－ｄ６） δ：１２．５６（ｂｒｓ， １Ｈ） ７．８５ （ｄ， Ｊ＝８．４ Ｈｚ， ２Ｈ）， ７．４１ （ｂｒｓ， １Ｈ）， ７．２７ （ｄ， Ｊ＝８．４ Ｈｚ， ２Ｈ）， ２．８５ （ｓ， ２Ｈ）， １．７７ （ｓ， ６Ｈ）， １．２３ （ｓ， ９Ｈ）。 Synthesis of 4-((3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentan-1-yl)methyl)benzoic acid (INX-SM-16-2)

procedure:
In a 100 mL single-neck round bottom flask, add methyl 4-((3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentan-1-yl)methyl)benzoate (INX-SM-16). -1) (0.9 g, 2.72 mmol) and THF:H2O (1:1, 6 mL). To this solution was added LiOH.H2O (0.65 g, 16.3 mmol) and stirred at 50 C for 5 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with 10% MeOH:DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a white solid. (0.8g, 92.82%). 1H NMR (DMSO-d6) δ: 12.56 (brs, 1H) 7.85 (d, J=8.4 Hz, 2H), 7.41 (brs, 1H), 7.27 (d, J= 8.4 Hz, 2H), 2.85 (s, 2H), 1.77 (s, 6H), 1.23 (s, 9H).

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＭＦ（２ｍＬ）中の４－（（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－イル）メチル）安息香酸（ＩＮＸ－ＳＭ－１６－２）（０．１８ｇ、０．５６ｍｍｏｌ）及び２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－３４－２）（０．２９ｇ、０．６８ｍｍｏｌ）を充填した。この溶液に、ＤＩＰＥＡ（０．８ｇ、１．４ｍｍｏｌ）及びＨＡＴＵ（０．３２ｇ、０．８５ｍｍｏｌ）を室温で添加し、１５分間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物をジエチルエーテルで粉砕して、オフホワイト色の固体として表題化合物を得た（０．１９ｇ、３８．５３％）。粗製の表題化合物をそのまま次のステップに進めた。ＬＣＭＳ：７２７．４３ ［Ｍ＋Ｈ］＋。 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-((3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentane-1 -yl)methyl)benzoyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b- Synthesis of tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-16-3)

procedure:
In a 10 mL single neck round bottom flask, add 4-((3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentan-1-yl)methyl)benzoic acid ( INX-SM-16-2) (0.18 g, 0.56 mmol) and 2-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-6a,8a-dimethyl-4 -Oxo-1,4,6a,6b,7,8,8a,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1, 2-c]pyrrole-8b(2H)-yl)-2-oxoethyl acetate (INX-SM-34-2) (0.29 g, 0.68 mmol) was charged. To this solution was added DIPEA (0.8 g, 1.4 mmol) and HATU (0.32 g, 0.85 mmol) at room temperature and stirred for 15 minutes. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was triturated with diethyl ether to give the title compound as an off-white solid (0.19 g, 38.53%). The crude title compound was carried on to the next step. LCMS: 727.43 [M+H]+.

手順：
１０ｍＬの一つ口丸底フラスコに、２－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（３－（（ｔｅｒｔ－ブトキシカルボニル）アミノ）ビシクロ［１．１．１］ペンタン－１－イル）メチル）ベンゾイル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－１，４，６ａ，６ｂ，７，８，８ａ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－テトラデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－８ｂ（２Ｈ）－イル）－２－オキソエチルアセテート（ＩＮＸ－ＳＭ－１６－３）（０．１９ｇ、０．２６ｍｍｏｌ）及びメタノール（１ｍＬ）を充填した。この溶液に、ＮａＨＣＯ３（０．０４ｇ、０．５３ｍｍｏｌ）を添加し、室温で１２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を酢酸エチルで希釈し、セライトベッドを通して濾過した。組み合わせた有機層を濃縮して、オフホワイト色の固体として表題化合物の粗製の生成物を得た（０．１７ｇ、粗製）。ＬＣＭＳ：５８５．２９ ［Ｍ＋Ｈ－Ｂｏｃ］＋。 (3-(4-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-1, 2,4,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-hexadecahydronaphtho[2',1':4,5]indeno[1,2 Synthesis of tert-butyl-c]pyrrole-10-carbonyl)benzyl)bicyclo[1.1.1]pentan-1-yl)carbamate (INX-SM-16-4)

procedure:
In a 10 mL single-neck round bottom flask, add 2-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-((tert-butoxycarbonyl)amino)bicyclo). [1.1.1] Pentan-1-yl)methyl)benzoyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,4,6a,6b,7,8,8a,9,10, 11,11a,12,12a,12b-tetradecahydronaphtho[2',1':4,5]indeno[1,2-c]pyrrol-8b(2H)-yl)-2-oxoethyl acetate (INX -SM-16-3) (0.19 g, 0.26 mmol) and methanol (1 mL) were charged. To this solution was added NaHCO3 (0.04 g, 0.53 mmol) and stirred at room temperature for 12 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ethyl acetate and filtered through a bed of Celite. The combined organic layers were concentrated to give the crude product of the title compound as an off-white solid (0.17 g, crude). LCMS: 585.29 [M+H-Boc]+.

手順：
１０ｍＬの一つ口丸底フラスコに、（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－１，２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，９，１０，１１，１１ａ，１２，１２ａ，１２ｂ－ヘキサデカヒドロナフト［２’，１’：４，５］インデノ［１，２－ｃ］ピロール－１０－カルボニル）ベンジル）ビシクロ［１．１．１］ペンタン－１－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１６－４）（０．２ｇ、０．２９ｍｍｏｌ）及びＤＣＭ（１ｍＬ）を充填した。この溶液に、ＴＦＡ（６０．４ｍｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＮＥＷ Ｘ－ｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ（２５０×１９）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル＋５％テトラヒドロフラン中の２０％Ａライン、Ａ：Ｂ＝７５：２５）により精製して、表題化合物（０．０２ｇ、１１．７１％）を得た。ＬＣＭＳ：５８５．５０ ［Ｍ＋Ｈ］＋；１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）：δ：７．５０－７．３９（ｍ， ３Ｈ）， ７．２３（ｄ， Ｊ＝７．６Ｈｚ， ２Ｈ）， ７．３０－７．２７（ｍ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ４．５７－３．５４（ｍ， ７Ｈ）， ２．９７（ｓ， ２Ｈ）， ２．８０－２．００（ｍ， ６Ｈ）， １．９０（ｓ， ６Ｈ）， １．８９－１．６０（ｍ， ２Ｈ）， １．５０（ｓ， ３Ｈ）， １．４５－１．１２（ｍ， ４Ｈ），１．１０（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-10-(4-((3-aminobicyclo[1.1.1]pentan-1-yl)methyl)benzoyl)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-tetradecahydronaphtho[2 Synthesis of ',1':4,5]indeno[1,2-c]pyrrol-4(2H)-one (INX-SM-16)

procedure:
In a 10 mL single-neck round bottom flask, add (3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a-Dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,8b,9,10,11,11a,12,12a,12b-hexadecahydronaphtho[2',1' :4,5]indeno[1,2-c]pyrrole-10-carbonyl)benzyl)bicyclo[1.1.1]pentan-1-yl)tert-butylcarbamate (INX-SM-16-4) ( 0.2 g, 0.29 mmol) and DCM (1 mL). TFA (60.4 ml) was added to this solution and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude material was subjected to preparative HPLC (column: NEW A line, A:B=75:25) to give the title compound (0.02 g, 11.71%). LCMS: 585.50 [M+H]+; 1H NMR (400 MHz, MeOD): δ: 7.50-7.39 (m, 3H), 7.23 (d, J=7.6Hz, 2H), 7 .30-7.27 (m, 1H), 6.04 (s, 1H), 4.57-3.54 (m, 7H), 2.97 (s, 2H), 2.80-2.00 (m, 6H), 1.90 (s, 6H), 1.89-1.60 (m, 2H), 1.50 (s, 3H), 1.45-1.12 (m, 4H), 1.10 (s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロブチル）（メチル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２１－１）の合成
ＩＮＸ－Ａ２１－１
手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．３４ｇ、１．６５ｍｍｏｌ）、ＨＡＴＵ（０．４０、１．０５ｍｍｏｌ）、ＤＩＰＥＡ（０．１８ｇ、１．４１ｍｍｏｌ）、及びＤＭＦ（３ｍＬ）を室温で充填した。この溶液に、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１０－（４－（（３－（メチルアミノ）シクロブチル）メチル）フェニル）－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－４９）（０．３９ｇ、０．７ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ２ＳＯ４上で乾燥させ、真空下で蒸発させた。粗生成物を逆相カラムクロマトグラフィー（アセトニトリル／水：６０：４０）によって精製して、薄黄色の固体として表題化合物を得た（０．３ｇ、４２．１０％）。ＬＣＭＳ：１０２６．９２ ［Ｍ＋Ｈ］＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl)(methyl)amino)-5-oxopentanoic acid (INX-A21) synthesis of

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS , 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a ,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl)(methyl)amino ) Synthesis of tert-butyl-5-oxopentanoate (INX-A21-1) INX-A21-1
procedure:
In a 50 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.34 g, 1.65 mmol), HATU (0.40, 1.05 mmol), DIPEA (0.18 g, 1.41 mmol), and DMF (3 mL) at room temperature. Filled. Add (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-10-(4-(( 3-(Methylamino)cyclobutyl)methyl)phenyl)-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4 ,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-49) (0.39 g, 0.7 mmol) was added and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under vacuum. The crude product was purified by reverse phase column chromatography (acetonitrile/water: 60:40) to give the title compound as a pale yellow solid (0.3 g, 42.10%). LCMS: 1026.92 [M+H]+.

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロブチル）（メチル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ－２１－１）（０．３ｇ、０．２９ｍｍｏｌ）及びＴＨＦ（４１００ｍＬ）を充填した。この溶液に、ジエチルアミン（０．２１ｇ、２．９ｍｍｏｌ）を室温で添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させて、黄色の固体として表題化合物を得た（０．１８ｇ、７６．５９％）ＬＣＭＳ：８０４．４１［Ｍ＋Ｈ］＋。 (S)-4-(2-aminoacetamide)-5-((3-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl)(methyl)amino)-tert-butyl-5-oxopentanoate (INX-A21-2) synthesis of

procedure:
In a 50 mL single neck round bottom flask, add (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-((3-(4-( (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b ,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole-10- tert-butyl)benzyl)cyclobutyl)(methyl)amino)-5-oxopentanoate (INX-A-21-1) (0.3 g, 0.29 mmol) and THF (4100 mL) were charged. Diethylamine (0.21 g, 2.9 mmol) was added to this solution at room temperature and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to give the title compound as a yellow solid (0.18 g, 76.59%) LCMS: 804.41 [M+H]+.

手順：
２５ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の（Ｓ）－４－（２－アミノアセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロブチル）（メチル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２１－２）（０．１８ｇ、０．２２ｍｍｏｌ）を充填した。この溶液に、水（０．５ｍＬ）中のＮａ_２ＣＯ_３（０．０４６ｇ、０．４４ｍｍｏｌ）溶液、続いてブロモアセチルブロミド（０．０６７ｇ、０．３３ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物をジエチルエーテルで粉砕して、淡黄色の固体として表題化合物を得た（０．１８ｇ、８６．９３％）。ＬＣＭＳ：Ｃ４７Ｈ６３７９ＢｒＦＮ３Ｏ１１に対する計算値（９２４．３６）、実測値９２４．７０［Ｍ＋Ｈ］＋。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho tert-butyl [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl)(methyl)amino)-5-oxopentanoate (INX -Synthesis of A21-3)

procedure:
In a 25 mL single neck round bottom flask, add (S)-4-(2-aminoacetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a, 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl)(methyl)amino) Tert-butyl-5-oxopentanoate (INX-A21-2) (0.18 g, 0.22 mmol) was charged. To this solution was added a solution of Na ₂ CO ₃ (0.046 g, 0.44 mmol) in water (0.5 mL) followed by bromoacetyl bromide (0.067 g, 0.33 mmol) at room temperature and stirred for 1 h. did. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was triturated with diethyl ether to give the title compound as a pale yellow solid (0.18 g, 86.93%). LCMS: Calcd for C47H6379BrFN3O11 (924.36), found 924.70 [M+H]+.

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（３－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロブチル）（メチル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２１－３）（０．１７ｇ、０．１８ｍｍｏｌ）を充填した。この溶液に、ＴＦＡ（０．１０ｇ、０．９１ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗生成物を分取ＨＰＬＣによって精製して、オフホワイト色の固体として表題化合物を得た（０．０２１ｇ、１３．１５％）；ＬＣＭＳ：Ｃ４３Ｈ５５７９ＢｒＦＮ３Ｏ１１に対する計算値（８６８．３０）、実測値８６８．４０［Ｍ＋Ｈ］＋；１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４６（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３８－７．３６（ｍ， ２Ｈ）， ７．２３－１９（ｍ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１０．０ ＆ ２．０Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５，４６（ｓ， アセタール－Ｈ， １Ｈ）， ５．０７ （ｓ， Ｊ＝５．２Ｈｚ， Ｃ１６－Ｈ， １Ｈ）， ５．０４－５．００（ｍ， １Ｈ）， ４．４６－４．１５（ｍ， ４Ｈ）， ３．９５－３．８９（ｍ， ４Ｈ）， ３．３０－１．６０（ｍ， ２３Ｈ）， １．５２（ｓ， ３Ｈ）， １．０８－１．０５（ｍ， ２Ｈ）１．００（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7- Hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl)(methyl)amino)-5-oxopentanoic acid (INX-A21) synthesis of

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide)-5-((3-(4-((6aR,6bS,7S)) in DCM (2 mL). , 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a ,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclobutyl) Tert-butyl (methyl)amino)-5-oxopentanoate (INX-A21-3) (0.17 g, 0.18 mmol) was charged. To this solution was added TFA (0.10 g, 0.91 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was purified by preparative HPLC to give the title compound as an off-white solid (0.021 g, 13.15%); LCMS: calcd for C43H5579BrFN3O11 (868.30), found 868. 40[M+H]+; 1H NMR (400 MHz, MeOD): δ: 7.46 (d, J=10.0Hz, 1H), 7.38-7.36 (m, 2H), 7.23-19 (m, 2H), 6.27 (dd, J=10.0 & 2.0Hz, 1H), 6.04 (s, 1H), 5,46 (s, acetal-H, 1H), 5.07 (s, J=5.2Hz, C16-H, 1H), 5.04-5.00 (m, 1H), 4.46-4.15 (m, 4H), 3.95-3.89 ( m, 4H), 3.30-1.60 (m, 23H), 1.52 (s, 3H), 1.08-1.05 (m, 2H) 1.00 (s, 3H).

５－（メトキシメチレン）ヘキサヒドロシクロペンタ［ｃ］ピロール－２（１Ｈ）－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４８－１）の合成

手順：
１００ｍＬの一つ口丸底フラスコに、５－オキソヘキサヒドロシクロペンタ［ｃ］ピロール－２（１Ｈ）－カルボン酸ｔｅｒｔ－ブチル（１．０ｇ、４．４４ｍｍｏｌ）、カリウムｔｅｒｔ－ブトキシド（０．９９ｇ、８．８８ｍｍｏｌ）、及びＴＨＦ（２０ｍＬ）を充填した。この溶液に、（メトキシメチル）トリフェニルホスホニウムクロリド（２．７４ｇ、７．９９ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を塩化アンモニウム溶液で希釈し、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、黄色の固体として表題化合物を得た（０．８ｇ、７１．１４％）、^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ－ｄ６）： δ：５．９８（ｓ， １Ｈ）， ３．４８（ｓ， ３Ｈ）， ３．４１－３．３７（ｍ， ２Ｈ）， ２．９８－２．９０（ｍ， ２Ｈ）， ２．５９－２．５３（ｍ， ２Ｈ）， ２．４１－２．３３（ｍ， ２Ｈ）， ２．０４－１．９６（ｍ， ２Ｈ）， １．３８（ｓ， ９Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-10-(4-((octahydrocyclopenta [c]pyrrol-5-yl)methyl)phenyl)-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1': Synthesis of 4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate) (INX-SM-48)

Synthesis of tert-butyl 5-(methoxymethylene)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (INX-SM-48-1)

procedure:
In a 100 mL single-necked round bottom flask, add tert-butyl 5-oxohexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (1.0 g, 4.44 mmol), potassium tert-butoxide (0.99 g). , 8.88 mmol), and THF (20 mL). To this solution was added (methoxymethyl)triphenylphosphonium chloride (2.74 g, 7.99 mmol) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with ammonium chloride solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a yellow solid (0.8 g, 71.14%), ¹ H NMR (400 MHz, DMSO- d6): δ: 5.98 (s, 1H), 3.48 (s, 3H), 3.41-3.37 (m, 2H), 2.98-2.90 (m, 2H), 2 .59-2.53 (m, 2H), 2.41-2.33 (m, 2H), 2.04-1.96 (m, 2H), 1.38 (s, 9H).

手順：
１００ｍＬの一つ口丸底フラスコに、アセトン（５ｍＬ）中の５－（メトキシメチレン）ヘキサヒドロシクロペンタ［ｃ］ピロール－２（１Ｈ）－カルボン酸メチルｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４８－１）（０．５５ｇ、２．１７ｍｍｏｌ）を充填した。この溶液に、ＰＴＳＡ（０．４１ｇ、２．１７ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＮａＨＣＯ_３水溶液でクエンチし、ジクロロメタンで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、ゴム状固体として表題化合物を得た（０．４５ｇ、９５．２８％）。粗製物を分析せずに次のステップに直接使用した。 Synthesis of tert-butyl 5-formylhexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (INX-SM-48-2)

procedure:
In a 100 mL single neck round bottom flask, add methyl tert-butyl 5-(methoxymethylene)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (INX-SM-48-1) in acetone (5 mL). ) (0.55 g, 2.17 mmol). To this solution was added PTSA (0.41 g, 2.17 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with aqueous NaHCO ₃ and extracted with dichloromethane. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound as a gummy solid (0.45 g, 95.28%). The crude material was used directly in the next step without analysis.

手順：
３５ｍＬのガラスバイアルに、５－ホルミルヘキサヒドロシクロペンタ［ｃ］ピロール－２（１Ｈ）－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４８－２）（０．４５ｇ、１．８８ｍｍｏｌ）及びエタノール（５ｍＬ）を充填した。この溶液に、ｐ－トルエンスルホンヒドラジド（０．３８ｇ、２．０６ｍｍｏｌ）及び酢酸（触媒）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、得られた白色の固体を濾過し、真空下で乾燥させて、表題化合物を得た（０．５８ｇ、７５．６９％）。Ｃ_２０Ｈ_３０Ｎ_３Ｏ_４Ｓに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、４０８．３であった。 (E) Synthesis of tert-butyl-5-((2-tosylhydrazono)methyl)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (INX-SM-48-3)

procedure:
In a 35 mL glass vial, tert-butyl 5-formylhexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (INX-SM-48-2) (0.45 g, 1.88 mmol) and ethanol (5 mL) were added. ) was filled. To this solution, p-toluenesulfone hydrazide (0.38 g, 2.06 mmol) and acetic acid (catalyst) were added and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and the resulting white solid was filtered and dried under vacuum to give the title compound (0.58 g, 75.69%). LCMS [M _{+ H} ] ⁺ observed value _{for C20H30N3O4S} was 408.3.

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｅ）－５－（（２－トシルヒドラゾノ）メチル）ヘキサヒドロシクロペンタ［ｃ］ピロール－２（１Ｈ）－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４８－３）（０．５８ｇ、１．４４ｍｍｏｌ）及びジオキサン（１０ｍＬ）を充填した。（４－ホルミルフェニル）ボロン酸（０．２１ｇ、１．４４ｍｍｏｌ）及びＫ_２ＣＯ_３（０．２９ｇ、２．１６ｍｍｏｌ）を添加し、反応混合物を１１０℃で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン、５０：５０）によって精製して、無色の液体として表題化合物を得た（０．１７ｇ、３５．８％）。Ｃ_１６Ｈ_２０ＮＯ_３に対するＬＣＭＳ［（Ｍ＋Ｈ）－^ｔＢｕ］^＋実測値は、２７３．８であった。 Synthesis of tert-butyl 5-(4-formylbenzyl)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (INX-SM-48-4)

procedure:
In a 50 mL single-necked round bottom flask, add tert-butyl (E)-5-((2-tosylhydrazono)methyl)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (INX-SM-48- 3) (0.58 g, 1.44 mmol) and dioxane (10 mL) were charged. (4-formylphenyl)boronic acid (0.21 g, 1.44 mmol) and K ₂ CO ₃ (0.29 g, 2.16 mmol) were added and the reaction mixture was stirred at 110° C. for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was purified by silica gel column chromatography (ethyl acetate/hexane, 50:50) to give the title compound as a colorless liquid (0.17 g, 35.8%). LCMS [(M+H) ^-tBu ] ⁺ observed value for C ₁₆ H ₂₀ NO ₃ was 273.8.

手順：
２５ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の５－（４－ホルミルベンジル）ヘキサヒドロシクロペンタ［ｃ］ピロール－２（１Ｈ）－カルボン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－４８－４）（０．１６ｇ、０．４８５ｍｍｏｌ）及び（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－アルファ－ヒドロキシプレドニゾロン）（０．１９ｇ、０．５１ｍｍｏｌ）を充填した。この溶液に、ＭｇＳＯ_４（０．３０ｇ、２．５５ｍｍｏｌ）及びＨＣｌＯ_４（０．２５ｇ、２．５５ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ ＡＱＵＡ（２５０×１９）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝６６：３４、保持時間８．７８分）により精製して、白色の固体として表題化合物を得た（０．０４０ｇ、１１．７４％）。Ｃ_３６Ｈ_４６ＮＯ_６に対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、５８８．５であった；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＤＭＳＯ－ｄ６）： δ：８．７６（ｂｒｓ， １Ｈ）， ８．５８（ｂｒｓ， １Ｈ）， ７．３７－７．３１（ｍ， ３Ｈ）， ７．２２－７．１６（ｍ， ２Ｈ）， ６．１７（ｄｄ， Ｊ＝１．６Ｈｚ及び１０．０ Ｈｚ， １Ｈ）， ５．９４（ｓ， １Ｈ）， ５．４１（ｓ， アセタール－Ｈ， １Ｈ）， ５．１２－５．１０（ｍ， １Ｈ）， ４．９２（ｄ， Ｊ＝４．８Ｈｚ， Ｃ１６－Ｈ， １Ｈ）， ４．８２（ｂｒｓ， １Ｈ）， ４．５３－４－４７（ｍ， １Ｈ）， ４．３０（ｂｒｓ， １Ｈ）， ４．２１－４．１７（ｍ， １Ｈ）， ３．２０－３．００（ｍ， ２Ｈ）， ２．８０－１．５１（ｍ， ２０Ｈ）， １．３８（ｓ， ３Ｈ）， １．０７－０．９６（ｍ， ２Ｈ）， ０．８７（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-10-(4-((octahydrocyclopenta [c]pyrrol-5-yl)methyl)phenyl)-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1': Synthesis of 4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-48)

procedure:
In a 25 mL single neck round bottom flask was added tert-butyl 5-(4-formylbenzyl)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (INX-SM-48-) in DCM (2 mL). 4) (0.16 g, 0.485 mmol) and (8S, 9S, 10R, 11S, 13S, 14S, 16R, 17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10, 13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-alpha-hydroxyprednisolone) ( 0.19 g, 0.51 mmol). To this solution were added MgSO ₄ (0.30 g, 2.55 mmol) and HClO ₄ (0.25 g, 2.55 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated _NaHCO3 solution and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was purified by preparative HPLC (column: YMC AQUA (250 x 19) mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 66:34, retention time 8.78 Purification (min.) gave the title compound as a white solid (0.040 g, 11.74%). LCMS [M+H] ⁺ actual value for C ₃₆ H ₄₆ NO ₆ was 588.5; ¹ H NMR (400 MHz, DMSO-d6): δ: 8.76 (brs, 1H), 8.58 ( brs, 1H), 7.37-7.31 (m, 3H), 7.22-7.16 (m, 2H), 6.17 (dd, J=1.6Hz and 10.0 Hz, 1H) , 5.94 (s, 1H), 5.41 (s, acetal-H, 1H), 5.12-5.10 (m, 1H), 4.92 (d, J=4.8Hz, C16- H, 1H), 4.82 (brs, 1H), 4.53-4-47 (m, 1H), 4.30 (brs, 1H), 4.21-4.17 (m, 1H), 3 .20-3.00 (m, 2H), 2.80-1.51 (m, 20H), 1.38 (s, 3H), 1.07-0.96 (m, 2H), 0.87 (s, 3H).

（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（５－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ヘキサヒドロシクロペンタ［ｃ］ピロール－２（１Ｈ）－イル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２０－１）の合成

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．５３ｇ、１．１０ｍｍｏｌ）、ＨＡＴＵ（０．６２、１．６４ｍｍｏｌ）、ＤＩＰＥＡ（０．２８ｇ、２．１９ｍｍｏｌ）、及びＤＭＦ（６ｍＬ）を室温で充填した。この溶液に、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１０－（４－（（オクタヒドロシクロペンタ［ｃ］ピロール－５－イル）メチル）フェニル）－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン２，２，２－トリフルオロアセテート（ＩＮＸ－ＳＭ－４８）（０．６４ｇ、０．９１ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物を逆相カラムクロマトグラフィー（アセトニトリル／水：６０：４０）によって精製して、淡黄色の固体として表題化合物を得た（０．３２ｇ、３３．３５％）。ＬＣＭＳ：Ｃ_６２Ｈ_７４Ｎ_３Ｏ_１２に対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、１０５２．５であった。 (4S)-4-(2-(2-bromoacetamide)acetamide)-5-(5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)hexahydrocyclopenta[c]pyrrole-2(1H)-l)-5- Synthesis of oxopentanoic acid (INX-A-20)

(4S)-4-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(5-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a, 12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)hexahydrocyclopenta[c] Synthesis of tert-butyl pyrrol-2(1H)-yl)-5-oxopentanoate (INX-A20-1)

procedure:
In a 50 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.53 g, 1.10 mmol), HATU (0.62, 1.64 mmol), DIPEA (0.28 g, 2.19 mmol), and DMF (6 mL) at room temperature. Filled. Add (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-10-(4-(( octahydrocyclopenta[c]pyrrol-5-yl)methyl)phenyl)-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-48) (0.64 g, 0.91 mmol ) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was purified by reverse phase column chromatography (acetonitrile/water: 60:40) to give the title compound as a pale yellow solid (0.32 g, 33.35%). LCMS _: LCMS [M+H] ⁺ observed value _{for C62H74N3O12} was 1052.5 _.

手順：
５０ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（５－（４（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ヘキサヒドロシクロペンタ［ｃ］ピロール－２（１Ｈ）－イル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２０－１）（０．３２ｇ、０．３０ｍｍｏｌ）及びＴＨＦ（３ｍＬ）を充填した。この溶液に、ジエチルアミン（０．２２ｇ、３．０４ｍｍｏｌ）を添加し、反応混合物を室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させて、黄色の固体として表題化合物を得た（０．２４ｇ、９６．４％）。Ｃ_４７Ｈ_６４Ｎ_３Ｏ_１０に対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、８３０．５であった。 (4S)-4-(2-aminoacetamide)-5-(5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2 -hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1' :4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-5-oxopentanoic acid tert- Synthesis of butyl (INX-A20-2)

procedure:
In a 50 mL single neck round bottom flask, add (4S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(5-(4((6aR) , 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7 , 8,8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl) Filled with tert-butyl)-hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-5-oxopentanoate (INX-A20-1) (0.32 g, 0.30 mmol) and THF (3 mL). did. To this solution was added diethylamine (0.22 g, 3.04 mmol) and the reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to give the title compound as a yellow solid (0.24 g, 96.4%). LCMS _[ M ₊ H] ⁺ observed value _{for C47H64N3O10} was 830.5.

手順：
２５ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の（４Ｓ）－４－（２－アミノアセトアミド）－５－（５－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ヘキサヒドロシクロペンタ［ｃ］ピロール－２（１Ｈ）－イル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２０－２）（０．２４ｇ、０．２８ｍｍｏｌ）を充填した。この溶液に、水（０．５ｍＬ）中のＮａ_２ＣＯ_３（０．０５８ｇ、０．５６ｍｍｏｌ）溶液、続いてブロモアセチルブロミド（０．０８７ｇ、０．４３ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物をジエチルエーテルで粉砕して、淡黄色の固体として表題化合物を得た（０．２０ｇ、７５．１％）。ＬＣＭＳ：Ｃ_４９Ｈ_６５ ^７９ＢｒＮ_３Ｏ_１１に対する計算値（９５０．３８）、実測値９５０．４０［Ｍ＋Ｈ］^＋ (4S)-4-(2-(2-bromoacetamide)acetamide)-5-(5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-5- Synthesis of tert-butyl oxopentanoate (INX-A20-3)

procedure:
In a 25 mL single neck round bottom flask, add (4S)-4-(2-aminoacetamide)-5-(5-(4-((6aR,6bS,7S,8aS,8bS,10R) in DCM (2 mL). , 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12 , 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)hexahydrocyclopenta[c]pyrrole tert-Butyl-2(1H)-yl)-5-oxopentanoate (INX-A20-2) (0.24 g, 0.28 mmol) was charged. To this solution was added a solution of Na ₂ CO ₃ (0.058 g, 0.56 mmol) in water (0.5 mL) followed by bromoacetyl bromide (0.087 g, 0.43 mmol) and stirred at room temperature for 1 h. did. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was triturated with diethyl ether to give the title compound as a pale yellow solid (0.20 g, 75.1%). LCMS: Calculated value for C ₄₉ H ₆₅ ⁷⁹ BrN ₃ O ₁₁ (950.38), actual value 950.40 [M+H] ⁺

手順：
１０ｍＬの一つ口丸底フラスコに、（４Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（５－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）ヘキサヒドロシクロペンタ［ｃ］ピロール－２（１Ｈ）－イル）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ２０－３）（０．１７ｇ、０．１８ｍｍｏｌ）及びＤＣＭ（２ｍＬ）を充填した。この溶液に、ＴＦＡ（０．１０ｇ、０．９１ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製の表題化合物を分取ＨＰＬＣ（ｎｅｗ Ｘｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ １９×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝９０：１０）により精製して、生成物の２つの異性体を得た。
Ｆｒ－１（保持時間：１８分）（０．００９２ｇ、５．７５％、白色の固体）ＬＣＭＳ：Ｃ_４５Ｈ_５７ ^７９ＢｒＮ_３Ｏ_１１に対する計算値（８９４．３２）， 実測値８９４．５ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４６（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３５（ｄ， Ｊ＝７．６Ｈｚ， ２Ｈ）， ７．１９（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２６（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４５（ｓ， １Ｈ， アセタール－Ｈ）， ５．０５（ｄ， Ｊ＝５．２Ｈｚ， １Ｈ）， ４．７６－４．４４（ｍ， ４Ｈ）， ３．９６－３．９２（ｍ， ４Ｈ）， ３．８０－３．４０ （ｍ， ４Ｈ）， ２．８０－１．６０（ｍ， ２０Ｈ）， １．５１（ｓ， ３Ｈ）， １．３１－１．０５（ｍ， ４Ｈ）， １．００（ｓ， ３Ｈ）。
Ｆｒ－２（保持時間：１９．５分）（０．００３５ｇ、２．１９％、白色の固体）。ＬＣＭＳ：Ｃ_４５Ｈ_５７ ^７９ＢｒＮ_３Ｏ_１１に対する計算値（８９４．３）、実測値８９４．３ ［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．３６（ｄ， Ｊ＝７．６Ｈｚ， ２Ｈ）， ７．２１－７．２０（ｍ， ２Ｈ）， ６．２７（ｄ， Ｊ＝１０Ｈｚ， １Ｈ）， ６．０５（ｓ， １Ｈ）， ５．４４（ｓ， １Ｈ， アセタール－Ｈ）， ５．０６（ｄ， Ｊ＝５．２Ｈｚ， １Ｈ）， ４．８０－４．３０（ｍ， ４Ｈ）， ４．００－３．７０（ｍ， ４Ｈ）， ３．６５－１．５ （ｍ， ２６Ｈ）， １．５４（ｓ， ３Ｈ）， １．２５－１．０５（ｍ， ２Ｈ）， １．００（ｓ， ３Ｈ）。 (4S)-4-(2-(2-bromoacetamide)acetamide)-5-(5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy -8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-5- Synthesis of oxopentanoic acid (INX-A20)

procedure:
In a 10 mL single-neck round bottom flask, (4S)-4-(2-(2-bromoacetamido)acetamide)-5-(5-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12, 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)hexahydrocyclopenta[c]pyrrole- tert-Butyl 2(1H)-yl)-5-oxopentanoate (INX-A20-3) (0.17 g, 0.18 mmol) and DCM (2 mL) were charged. To this solution was added TFA (0.10 g, 0.91 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude title compound was purified by preparative HPLC (new Xbridge Prep, C18, OBD 19 x 250 mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 90:10). Two isomers of the product were obtained.
Fr-1 (retention time: 18 min) (0.0092 g, 5.75%, white solid) LCMS: Calcd for C ₄₅ H ₅₇ ⁷⁹ BrN ₃ O ₁₁ (894.32), found 894.5 [ M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ: 7.46 (d, J=10.0 Hz, 1H), 7.35 (d, J=7.6 Hz, 2H), 7.19 ( d, J=8.0Hz, 2H), 6.26(d, J=10Hz, 1H), 6.04(s, 1H), 5.45(s, 1H, acetal-H), 5.05( d, J=5.2Hz, 1H), 4.76-4.44 (m, 4H), 3.96-3.92 (m, 4H), 3.80-3.40 (m, 4H), 2.80-1.60 (m, 20H), 1.51 (s, 3H), 1.31-1.05 (m, 4H), 1.00 (s, 3H).
Fr-2 (retention time: 19.5 minutes) (0.0035 g, 2.19%, white solid). LCMS: Calculated value for C ₄₅ H ₅₇ ⁷⁹ BrN ₃ O ₁₁ (894.3), actual value 894.3 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ: 7.47 (d, J= 10.0Hz, 1H), 7.36 (d, J=7.6Hz, 2H), 7.21-7.20 (m, 2H), 6.27 (d, J=10Hz, 1H), 6. 05 (s, 1H), 5.44 (s, 1H, acetal-H), 5.06 (d, J=5.2Hz, 1H), 4.80-4.30 (m, 4H), 4. 00-3.70 (m, 4H), 3.65-1.5 (m, 26H), 1.54 (s, 3H), 1.25-1.05 (m, 2H), 1.00 ( s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（（１Ｓ，４Ｒ）－４－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロヘキシル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ－２８－１）の合成

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．３９ｇ、０．８１ｍｍｏｌ）、ＨＡＴＵ（０．３７ｇ、０．９２４ｍｍｏｌ）、ＤＩＰＥＡ（０．２６ｇ、０．８２ｍｍｏｌ）、及びＤＭＦ（５ｍＬ）を室温で充填した。この溶液に、ｔｅｒｔ－ブチル（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（（１ｓ，４Ｓ）－４－アミノシクロヘキシル）メチル）フェニル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－１５）（０．５ｇ、０．８１ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物を逆相カラムクロマトグラフィー（アセトニトリル／水：５０：５０）によって精製して、薄黄色の固体として表題化合物を得た（０．４５ｇ、４１．８％）。Ｃ_６１Ｈ_７２Ｆ_２Ｎ_３Ｏ_１２に対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、１０７７．０［Ｍ＋１］^＋であった。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-(((1S,4R)-4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR , 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b , 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclohexyl)amino) Synthesis of -5-oxopentanoic acid (INX-A-28)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(((1S,4R)-4-(4-((2S,6aS) , 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4 , 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3] Synthesis of tert-butyl dioxol-10-yl)benzyl)cyclohexyl)amino)-5-oxopentanoate (INX-A-28-1)

procedure:
In a 10 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.39 g, 0.81 mmol), HATU (0.37 g, 0.924 mmol), DIPEA (0.26 g, 0.82 mmol), and DMF (5 mL) at room temperature. Filled. Add tert-butyl (2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-(((1s,4S)-4-aminocyclohexyl)methyl)phenyl to this solution. )-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b -dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-15) (0.5 g, 0.81 mmol ) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was purified by reverse phase column chromatography (acetonitrile/water: 50:50) to give the title compound as a pale yellow solid (0.45 g, 41.8%). The LCMS [M+H] ⁺ actual value for C ₆₁ H ₇₂ F ₂ N ₃ O ₁₂ was 1077.0 [M+1] ⁺ .

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（（１Ｓ，４Ｒ）－４－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロヘキシル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ－２８－１）（０．４ｇ、０．３７ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ジエチルアミン（０．２７ｇ、３．７２ｍｍｏｌ）を添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、粗生成物をジエチルエーテル及びペンタンで粉砕して、黄色の固体として表題化合物を得た（０．２５ｇ、７９．１％）。Ｃ_４６Ｈ_６２Ｆ_２Ｎ_３Ｏ_１０に対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、８５４．８であった。 (S)-4-(2-aminoacetamide)-5-(((1S,4R)-4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS) -2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12, 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclohexyl)amino)-5-oxopentane Synthesis of tert-butyl acid (INX-A-28-2)

procedure:
In a 10 mL single neck round bottom flask, (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(((1S,4R)- 4-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a -dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1 ,2-d][1,3]dioxol-10-yl)benzyl)cyclohexyl)amino)-5-oxopentanoate tert-butyl (INX-A-28-1) (0.4 g, 0.37 mmol) and Charged with THF (5 mL). To this solution was added diethylamine (0.27 g, 3.72 mmol) and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and the crude product was triturated with diethyl ether and pentane to give the title compound as a yellow solid (0.25 g, 79.1%) . LCMS [M+H] ⁺ observed value for C ₄₆ H ₆₂ F ₂ N ₃ O ₁₀ was 854.8.

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－アミノアセトアミド）－５－（（（１Ｓ，４Ｒ）－４－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロヘキシル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ－２８－２）（０．２５ｇ、０．２９ｍｍｏｌ）及びＤＣＭ（４ｍＬ）を充填した。この溶液に、水（０．５ｍＬ）中に溶解したＮａ_２ＣＯ_３（０．１２ｇ、１．１７ｍｍｏｌ）、続いてブロモアセチルブロミド（０．１１７ｇ、０．５８ｍｍｏｌ）を反応混合物に添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物を逆相カラムクロマトグラフィー（アセトニトリル／水：５０：５０）によって精製して、淡黄色の固体として表題化合物を得た（０．２ｇ、７０．０８％）。ＬＣＭＳ：Ｃ_４８Ｈ_６３ ^７９ＢｒＦ_２Ｎ_３Ｏ_１１に対する計算値（９７４．３６）、実測値９７４．４［Ｍ＋Ｈ］^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-(((1S,4R)-4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR , 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b , 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclohexyl)amino) Synthesis of tert-butyl-5-oxopentanoate (INX-A-28-3)

procedure:
In a 10 mL single neck round bottom flask, (S)-4-(2-aminoacetamide)-5-(((1S,4R)-4-(4-((2S,6aS,6bR,7S,8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7, 8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl ) cyclohexyl)amino)-tert-butyl-5-oxopentanoate (INX-A-28-2) (0.25 g, 0.29 mmol) and DCM (4 mL) were charged. To this solution, Na ₂ CO ₃ (0.12 g, 1.17 mmol) dissolved in water (0.5 mL) followed by bromoacetyl bromide (0.117 g, 0.58 mmol) were added to the reaction mixture and the reaction mixture was heated to room temperature. The mixture was stirred for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was purified by reverse phase column chromatography (acetonitrile/water: 50:50) to give the title compound as a pale yellow solid (0.2 g, 70.08%). LCMS: Calculated value for C ₄₈ H ₆₃ ⁷⁹ BrF ₂ N ₃ O ₁₁ (974.36), actual value 974.4 [M+H] ⁺

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＣＭ（４ｍＬ）中の（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（（１Ｓ，４Ｒ）－４－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロヘキシル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ－２８－３）（０．２ｇ、０．２０ｍｍｏｌ）を充填した。この溶液に、ＴＦＡ（０．４ｍＬ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：Ｘ－ｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ（２５０×１９）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝６５：３５、保持時間１４．４０分）により精製して、白色の固体として表題化合物を得た（０．０１５ｇ、８．１６％）。ＬＣＭＳ：Ｃ_４４Ｈ_５５ ^７９ＢｒＦ_２Ｎ_３Ｏ_１１に対する計算値（９１８．３０）、実測値９１８．５［Ｍ＋Ｈ］^＋；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＣＤ３ＯＤ）： δ：７．４５－７．３６（ｍ， ３Ｈ）， ７．２２－７．２０（ｍ， ２Ｈ）， ６．４０－６．３５（ｍ， ２Ｈ）， ５．６６－５．５０（ｍ， １Ｈ， ＣＨ－Ｆ）， ５．４８（ｓ， １Ｈ， アセタール－Ｈ）， ５．０７（ｄ， Ｊ＝４．４Ｈｚ， １Ｈ）， ４．６６ （ｄ， １Ｈ）， ４．４３－４．３０（ｍ， ３Ｈ）， ３．９５（ｓ， ２Ｈ）， ３．９２（ｓ， ２Ｈ）， ３．８６（ｂｒｓ， １Ｈ）， ２．８０－１．６５（ｍ， １７Ｈ）， １．５５（ｓ， ３Ｈ）， １．５０－１．３６（ｍ， ６Ｈ）， １．０１（ｓ， ３Ｈ）。 (S)-4-(2-(2-bromoacetamide)acetamide)-5-(((1S,4R)-4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR , 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b , 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclohexyl)amino) Synthesis of -5-oxopentanoic acid (INX-A-28)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide)-5-(((1S,4R)-4-(4-( (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ Tert-butyl 1,3]dioxol-10-yl)benzyl)cyclohexyl)amino)-5-oxopentanoate (INX-A-28-3) (0.2 g, 0.20 mmol) was charged. To this solution was added TFA (0.4 mL) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: , retention time 14.40 min) to give the title compound as a white solid (0.015 g, 8.16%). LCMS: Calculated value for C ₄₄ H ₅₅ ⁷⁹ BrF ₂ N ₃ O ₁₁ (918.30), actual value 918.5 [M+H] ⁺ ; ¹ H NMR (400 MHz, CD3OD): δ: 7.45-7. 36 (m, 3H), 7.22-7.20 (m, 2H), 6.40-6.35 (m, 2H), 5.66-5.50 (m, 1H, CH-F), 5.48 (s, 1H, acetal-H), 5.07 (d, J=4.4Hz, 1H), 4.66 (d, 1H), 4.43-4.30 (m, 3H), 3.95 (s, 2H), 3.92 (s, 2H), 3.86 (brs, 1H), 2.80-1.65 (m, 17H), 1.55 (s, 3H), 1 .50-1.36 (m, 6H), 1.01 (s, 3H).

（４－ヒドロキシシクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１８－１）の合成

手順：
１００ｍＬの一つ口丸底フラスコに、（４－オキソシクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（５．０ｇ、２３．４３ｍｍｏｌ）及びＭｅＯＨ（５０ｍＬ）を窒素下で充填した。この溶液に、ＮａＢＨ_４（７．４３ｇ、１１７．１６ｍｍｏｌ）を０℃で少しずつ添加し、０℃で更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水で希釈し、１Ｎ ＨＣｌで中性ｐＨに調整し、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させて、表題化合物（４．８ｇ、９５．１０％）を得た。Ｃ_１１Ｈ_２２ＮＯ_３に対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、２１６．２であった。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((4-aminocyclohexyl)oxy)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)- 6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1, Synthesis of 2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-18)

Synthesis of tert-butyl (4-hydroxycyclohexyl)carbamate (INX-SM-18-1)

procedure:
A 100 mL single neck round bottom flask was charged with tert-butyl (4-oxocyclohexyl)carbamate (5.0 g, 23.43 mmol) and MeOH (50 mL) under nitrogen. NaBH ₄ (7.43 g, 117.16 mmol) was added portionwise to this solution at 0° C. and stirred for an additional hour at 0° C. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with water, adjusted to neutral pH with 1N HCl, and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum to give the title compound (4.8 g, 95.10%). LCMS [M ₊ H] ⁺ observed value _{for C11H22NO3} was 216.2.

手順：
３５ｍＬのガラスバイアルに、ＴＨＦ（１０ｍＬ）中の（４－ヒドロキシシクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１８－１）（１ｇ、４．６４ｍｍｏｌ）、４－ヒドロキシベンズアルデヒド（０．６８０ｇ、５．５７ｍｍｏｌ）、及びトリフェニルホスフィン（１．８２ｇ、６．９６ｍｍｏｌ）を窒素下で充填した。この溶液にＤＩＡＤ（１．４ｇ、６．９６７ｍｍｏｌ）を０℃で添加し、反応混合物を６５℃で１６時間還流した。ＴＬＣによって示される反応の完了後、反応混合物を１０％ＮａＯＨ溶液で希釈し、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物をカラムクロマトグラフィー（酢酸エチル／ヘキサン、８０：２０）によって精製して、白色の固体として表題化合物を得た（１．０ｇ、６７．４０％）。Ｃ_１８Ｈ_２６ＮＯ_４に対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、３２０．２であった。 Synthesis of tert-butyl (4-(4-formylphenoxy)cyclohexyl)carbamate (INX-SM-18-2)

procedure:
In a 35 mL glass vial, tert-butyl (4-hydroxycyclohexyl)carbamate (INX-SM-18-1) (1 g, 4.64 mmol), 4-hydroxybenzaldehyde (0.680 g, 5 .57 mmol) and triphenylphosphine (1.82 g, 6.96 mmol) under nitrogen. DIAD (1.4 g, 6.967 mmol) was added to this solution at 0°C and the reaction mixture was refluxed at 65°C for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with 10% NaOH solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was purified by column chromatography (ethyl acetate/hexanes, 80:20) to give the title compound as a white solid (1.0 g, 67.40%). LCMS [M ₊ H] ⁺ observed value _{for C18H26NO4} was 320.2.

手順：
５０ｍＬの一つ口丸底フラスコに、ＤＣＭ（１０ｍＬ）中の（４－（４－ホルミルフェノキシ）シクロヘキシル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－１８－２）（１ｇ、３．１３ｍｍｏｌ）、（８Ｓ，９Ｓ，１０Ｒ，１１Ｓ，１３Ｓ，１４Ｓ，１６Ｒ，１７Ｓ）－１１，１６，１７－トリヒドロキシ－１７－（２－ヒドロキシアセチル）－１０，１３－ジメチル－６，７，８，９，１０，１１，１２，１３，１４，１５，１６，１７－ドデカヒドロ－３Ｈ－シクロペンタ［ａ］フェナントレン－３－オン（１６－アルファ－ヒドロキシプレドニゾロン）（１．１７ｇ、３．１３ｍｍｏｌ）を充填し、この溶液に、ＭｇＳＯ_４（１．５７ｇ、１５．６５ｍｍｏｌ）及びＨＣｌＯ_４（１．８８ｇ、１５．６５ｍｍｏｌ）を添加し、室温で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液を真空下で濃縮した。粗製物を冷水で粉砕し、１．５ｇの粗製の固体を得た。粗製物の一部（３０８ｍｇ）を分取ＨＰＬＣ（カラム：ＳＨＩＭ－ＰＡＣＫ－ＧＩＳＴ Ｃ１８（２５０８（２５０×２０）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝７０：３０、保持時間１１．８０分）により精製して、精製した総質量０．０９２ｇ（０．１３７ｍｍｏｌ、４．３８％）に相当する０．０１９ｇの表題化合物を白色の固体として得た。Ｃ_３４Ｈ_４３ＮＯ_７に対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、５７８．４であった；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４７（ｄ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ７．４０－７．３６（ｍ， ２Ｈ），６．９８－６．９２（ｍ， ２Ｈ）， ６．２７（ｄｄ， Ｊ＝１．６Ｈｚ， Ｊ＝１０．０Ｈｚ， １Ｈ）， ６．０４（ｓ， １Ｈ）， ５．４３（ｍ， １Ｈ）， ５．０４（ｄ， Ｊ＝５．２Ｈｚ， １Ｈ）， ４．６５（ｄ， １Ｈ）， ４．４４（ｂｒｓ， １Ｈ）， ４．３６－４．３０（ｍ， ２Ｈ）， ３．２０（ｂｒｓ， １Ｈ）， ２．８０－１．５４（ｍ， １５Ｈ）， １．４９（ｓ， ３Ｈ）， １．３０－１．０２（ｍ， ４Ｈ）， １．００（ｓ， ３Ｈ）。 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((4-aminocyclohexyl)oxy)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)- 6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1, Synthesis of 2-d][1,3]dioxol-4-one 2,2,2-trifluoroacetate (INX-SM-18)

procedure:
In a 50 mL single neck round bottom flask, tert-butyl (4-(4-formylphenoxy)cyclohexyl)carbamate (INX-SM-18-2) (1 g, 3.13 mmol) in DCM (10 mL), ( 8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10 , 11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (16-alpha-hydroxyprednisolone) (1.17 g, 3.13 mmol). MgSO ₄ (1.57 g, 15.65 mmol) and HClO ₄ (1.88 g, 15.65 mmol) were added to the solution and stirred for an additional 2 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated under vacuum with saturated _NaHCO3 solution. The crude was triturated with cold water to obtain 1.5 g of crude solid. A portion of the crude material (308 mg) was purified by preparative HPLC (column: SHIM-PACK-GIST C18 (2508 (250 x 20) mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A :B=70:30, retention time 11.80 min) to give 0.019 g of the title compound as a white solid, corresponding to a total purified mass of 0.092 g (0.137 mmol, 4.38%). LCMS [M+H] ⁺ actual value for C ₃₄ H ₄₃ NO ₇ was 578.4; ¹ H NMR (400 MHz, MeOD): δ: 7.47 (d, J=10.0 Hz, 1H), 7.40-7.36 (m, 2H), 6.98-6.92 (m, 2H), 6.27 (dd, J=1.6Hz, J=10.0Hz, 1H), 6.04 (s, 1H), 5.43 (m, 1H), 5.04 (d, J=5.2Hz, 1H), 4.65 (d, 1H), 4.44 (brs, 1H) , 4.36-4.30 (m, 2H), 3.20 (brs, 1H), 2.80-1.54 (m, 15H), 1.49 (s, 3H), 1.30-1 .02 (m, 4H), 1.00 (s, 3H).

（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（（１Ｒ，４Ｓ）－４－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロヘキシル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ－２７－１）の合成

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－２－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（ｔｅｒｔ－ブトキシ）－５－オキソペンタン酸（ＩＮＸ－Ｐ－４）（０．３９ｇ、０．８１ｍｍｏｌ）、ＨＡＴＵ（０．４６、１．２ｍｍｏｌ）、ＤＩＰＥＡ（０．２０ｇ、１．６２ｍｍｏｌ）、及びＤＭＦ（３ｍＬ）を室温で充填した。この溶液に、（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（（１ｒ，４Ｒ）－４－アミノシクロヘキシル）メチル）フェニル）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（ＩＮＸ－ＳＭ－１４）（０．４９ｇ、０．８１ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物を逆相カラムクロマトグラフィー（アセトニトリル／水：６０：４０）によって精製して、オフホワイト色の固体として表題化合物を得た（０．５０ｇ、５７．４８％）。ＬＣＭＳ：１０７６．７ ［Ｍ＋Ｈ］^＋ S)-4-(2-(2-bromoacetamide)acetamide)-5-(((1R,4S)-4-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclohexyl)amino)- Synthesis of 5-oxopentanoic acid (INX-A27)

(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(((1R,4S)-4-(4-((2S,6aS) , 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4 , 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3] Synthesis of tert-butyl dioxol-10-yl)benzyl)cyclohexyl)amino)-5-oxopentanoate (INX-A-27-1)

procedure:
In a 50 mL single neck round bottom flask, (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5- Oxopentanoic acid (INX-P-4) (0.39 g, 0.81 mmol), HATU (0.46, 1.2 mmol), DIPEA (0.20 g, 1.62 mmol), and DMF (3 mL) at room temperature. Filled. Add (2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-(((1r,4R)-4-aminocyclohexyl)methyl)phenyl)-2 to this solution. ,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- Add 4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (INX-SM-14) (0.49 g, 0.81 mmol) The mixture was stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was poured into water and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was purified by reverse phase column chromatography (acetonitrile/water: 60:40) to give the title compound as an off-white solid (0.50 g, 57.48%). LCMS: 1076.7 [M+H] ⁺

手順：
５０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（（（（９Ｈ－フルオレン－９－イル）メトキシ）カルボニル）アミノ）アセトアミド）－５－（（（１Ｒ，４Ｓ）－４－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロヘキシル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ－２７－１）（０．５ｇ、０．４６ｍｍｏｌ）及びＴＨＦ（５ｍＬ）を充填した。この溶液に、ジエチルアミン（０．３２ｇ、４．６ｍｍｏｌ）を添加し、室温で３時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させて、黄色の固体として表題化合物を得た（０．３ｇ、７５．６１％）ＬＣＭＳ：８５４．６４ ［Ｍ＋Ｈ］^＋ (S)-4-(2-aminoacetamide)-5-(((1R,4S)-4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS) -2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12, 12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclohexyl)amino)-5-oxopentane Synthesis of tert-butyl acid (INX-A-27-2)

procedure:
In a 50 mL single neck round bottom flask, (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(((1R,4S)- 4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a -dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1 ,2-d][1,3]dioxol-10-yl)benzyl)cyclohexyl)amino)-5-oxopentanoate tert-butyl (INX-A-27-1) (0.5 g, 0.46 mmol) and Charged with THF (5 mL). To this solution was added diethylamine (0.32 g, 4.6 mmol) and stirred at room temperature for 3 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum to give the title compound as a yellow solid (0.3 g, 75.61%) LCMS: 854.64 [M+H] ⁺

手順：
２５ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の（Ｓ）－４－（２－アミノアセトアミド）－５－（（（１Ｒ，４Ｓ）－４－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロヘキシル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ－２７－２）（０．３ｇ、０．３５ｍｍｏｌ）を充填した。この溶液に、水（０．５ｍＬ）中のＮａ_２ＣＯ_３（０．０７３ｇ、０．７ｍｍｏｌ）溶液、続いてブロモアセチルブロミド（０．１０ｇ、０．５２ｍｍｏｌ）を室温で添加し、１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を水でクエンチし、ＤＣＭで抽出した。合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗生成物をジエチルエーテルで粉砕して、淡黄色の固体として表題化合物を得た（０．３０ｇ、８７．６０％）。ＬＣＭＳ：Ｃ_４８Ｈ_６３ ^７９ＢｒＦ_２Ｎ_３Ｏ_１１に対する計算値（９７４．３６）、実測値９７４．６９ ［Ｍ＋Ｈ］^＋ (S)-4-(2-(2-bromoacetamide)acetamide)-5-(((1R,4S)-4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR , 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b , 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclohexyl)amino) Synthesis of tert-butyl-5-oxopentanoate (INX-A-27-3)

procedure:
In a 25 mL single neck round bottom flask, add (S)-4-(2-aminoacetamido)-5-(((1R,4S)-4-(4-((2S,6aS, 6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4, 6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole tert-Butyl-10-yl)benzyl)cyclohexyl)amino)-5-oxopentanoate (INX-A-27-2) (0.3 g, 0.35 mmol) was charged. To this solution was added a solution of Na ₂ CO ₃ (0.073 g, 0.7 mmol) in water (0.5 mL) followed by bromoacetyl bromide (0.10 g, 0.52 mmol) at room temperature and stirred for 1 h. did. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with water and extracted with DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was triturated with diethyl ether to give the title compound as a pale yellow solid (0.30 g, 87.60%). LCMS: Calculated value for C ₄₈ H ₆₃ ⁷⁹ BrF ₂ N ₃ O ₁₁ (974.36), actual value 974.69 [M+H] ⁺

手順：
１０ｍＬの一つ口丸底フラスコに、ＤＣＭ（２ｍＬ）中の（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（（１Ｒ，４Ｓ）－４－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）シクロヘキシル）アミノ）－５－オキソペンタン酸ｔｅｒｔ－ブチル（ＩＮＸ－Ａ－２７－３）（０．１５ｇ、０．１５ｍｍｏｌ）を充填した。この溶液に、ＴＦＡ（０．０８７ｇ、０．７６ｍｍｏｌ）を添加し、室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：ＹＭＣ－Ａｃｔｕｓ Ｔｒｉａｒｔ Ｐｒｅｐ Ｃ１８－Ｓ、２５０×２０ｍｍ Ｓ－１０μｍ、１２ｎｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝６０：４０、保持時間１６．００分）により精製して、白色固体として表題化合物を得た（０．０１０ｇ、７．０７％）、ＬＣＭＳ：Ｃ_４４Ｈ_５５ ^７９ＢｒＦ_２Ｎ_３Ｏ_１１に対する計算値（９１８．３０）， 実測値９１８．２０ ［Ｍ＋Ｈ］^＋ ；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．３７－７．３３（ｍ， ３Ｈ）， ７．１７（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．３８－６．３５（ｍ， ２Ｈ）， ５．６６－５．５０（ｍ， １Ｈ， ＣＨ－Ｆ）， ５．４８（ｓ， １Ｈ）， ５．０５（ｄ， Ｊ＝４．８Ｈｚ， １Ｈ）， ４．６５（ｄ， １Ｈ）， ４．３７－４．３１（ｍ， ３Ｈ）， ３．９４ （ｓ， ２Ｈ）， ３．９０（ｓ， ２Ｈ）， ３．６０－３．５０（ｍ， １Ｈ）， ２．８０－１．６６（ｍ， １８Ｈ）， １．６２（ｓ， ３Ｈ）， １．５０－１．０４（ｍ， ５Ｈ）， １．００（ｓ， ３Ｈ） (S)-4-(2-(2-bromoacetamide)acetamide)-5-(((1R,4S)-4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR , 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b , 11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)cyclohexyl)amino) Synthesis of -5-oxopentanoic acid (INX-A-27)

procedure:
In a 10 mL single neck round bottom flask, add (S)-4-(2-(2-bromoacetamido)acetamide)-5-(((1R,4S)-4-(4-( (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ Tert-butyl 1,3]dioxol-10-yl)benzyl)cyclohexyl)amino)-5-oxopentanoate (INX-A-27-3) (0.15 g, 0.15 mmol) was charged. To this solution was added TFA (0.087 g, 0.76 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: YMC-Actus Triart Prep C18-S, 250 x 20 mm S-10 μm, 12 nm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 60: 40, _{retention time} 16.00 min) to give the title compound as _a white solid (0.010 g, ^7.07 %), LCMS: calcd for _{C44H5579BrF2N3O 11} ( 918.30), actual value 918.20 [M+H] ⁺ ; ¹ H NMR (400 MHz, MeOD): δ: 7.37-7.33 (m, 3H), 7.17 (d, J=8. 0Hz, 2H), 6.38-6.35 (m, 2H), 5.66-5.50 (m, 1H, CH-F), 5.48 (s, 1H), 5.05 (d, J=4.8Hz, 1H), 4.65 (d, 1H), 4.37-4.31 (m, 3H), 3.94 (s, 2H), 3.90 (s, 2H), 3 .60-3.50 (m, 1H), 2.80-1.66 (m, 18H), 1.62 (s, 3H), 1.50-1.04 (m, 5H), 1.00 (s, 3H)

（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３８－１）の合成

手順：
１００ｍＬの一つ口丸底フラスコに、（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－１０－（４－（（６－アミノスピロ［３．３］ヘプタン－２－イル）メチル）フェニル）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（１．０ｇ、１．７０ｍｍｏｌ）（ＩＮＸ－ＳＭ－３２）、ＴＥＡ（０．４７ｍＬ、３．４０ｍｍｏｌ）、及びＤＣＭ（１０ｍＬ）－ＭｅＯＨ（０．５ｍＬ）を室温で充填した。この溶液に、二炭酸ジ－ｔｅｒｔ－ブチル（０．４７ｇ、３．４０ｍｍｏｌ）を添加し、反応混合物を室温で２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で直接蒸発させた。粗生成物を逆相カラムクロマトグラフィー（アセトニトリル：水、７０：３０）によって精製して、淡黄色の固体として表題化合物を得た（０．２５ｇ、２１．３６％）。Ｃ_４１Ｈ_５４ＮＯ_８に対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、６８８．５［Ｍ＋Ｈ］^＋であった。 6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl) -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ Synthesis of 1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane-2-aminium 2,2,2-trifluoroacetate (INX-SM-38)

(6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo- 2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1 , 3] Synthesis of tert-butyl dioxol-10-yl) benzyl) spiro[3.3] heptan-2-yl) carbamate (INX-SM-38-1)

procedure:
In a 100 mL single-neck round bottom flask, add (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl) ) methyl) phenyl) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro -4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one (1.0 g, 1.70 mmol) (INX-SM-32), TEA (0.47 mL, 3.40 mmol) and DCM (10 mL)-MeOH (0.5 mL) were charged at room temperature. To this solution was added di-tert-butyl dicarbonate (0.47 g, 3.40 mmol) and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction as indicated by TLC, the reaction mixture was directly evaporated under vacuum. The crude product was purified by reverse phase column chromatography (acetonitrile:water, 70:30) to give the title compound as a pale yellow solid (0.25g, 21.36%). LCMS [M+H] ⁺ observed value for C ₄₁ H ₅₄ NO ₈ was 688.5 [M+H] ⁺ .

手順：
３５ｍＬのガラスバイアルに、ＤＭＦ（０．５ｍＬ）中の（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（０．２５ｇ、０．３６ｍｍｏｌ）（ＩＮＸ－ＳＭ－３８－１）を充填した。この溶液に、１Ｈ－テトラゾール（０．２５４ｇ、３．６３ｍｍｏｌ）及び（ｔＢｕＯ）_２ＰＮＥｔ_２（２．４２ｇ、８．７３ｍｍｏｌ）を添加し、室温で２４時間撹拌した。過酸化水素（２．５ｍＬ）を反応混合物に－５～０℃で添加し、室温で１時間撹拌した。粗生成物を逆相カラムクロマトグラフィー（アセトニトリル：水、９０：１０）によって精製して、オフホワイト色の固体として表題化合物を得た（０．１８ｇ、５６．８１％）。Ｃ_４９Ｈ_７１ＮＯ_１１Ｐに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、８８０．６［Ｍ＋Ｈ］^＋であった。 (6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy- 6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5] Synthesis of tert-butyl indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-38-2)

procedure:
In a 35 mL glass vial, (6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1': tert-butyl)indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)carbamate (0.25 g, 0.36 mmol ) (INX-SM-38-1). To this solution were added 1H-tetrazole (0.254 g, 3.63 mmol) and (tBuO) ₂ PNEt ₂ (2.42 g, 8.73 mmol) and stirred at room temperature for 24 hours. Hydrogen peroxide (2.5 mL) was added to the reaction mixture at −5 to 0° C. and stirred at room temperature for 1 hour. The crude product was purified by reverse phase column chromatography (acetonitrile:water, 90:10) to give the title compound as an off-white solid (0.18g, 56.81%). The LCMS [M+H] ⁺ observed value for C ₄₉ H ₇₁ NO ₁₁ P was 880.6 [M+H] ⁺ .

手順：
２５ｍｌの一つ口丸底フラスコに、ＤＣＭ（４ｍＬ）中の（６－（４－（（６ａＲ，６ｂＳ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－８ｂ－（２－（（ジ－ｔｅｒｔ－ブトキシホスホリル）オキシ）アセチル）－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（０．１７ｇ、０．１９３ｍｍｏｌ）（ＩＮＸ－ＳＭ－３８－２）を充填した。この溶液に、ＴＦＡ（０．１７ｍＬ）を０℃で添加し、更に１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：Ｎｅｗ Ｘ－ｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ １９×２５０ｍｍ、５μｍ 移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝７２：２８、保持時間１１．７分）により精製して、白色の固体として表題化合物を得た（０．０２５ｇ、１６．９３％）。Ｃ_３６Ｈ_４７ＮＯ_９Ｐに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、６６８．４であった；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．４７（ｄｄ， Ｊ＝１０ ＆ ２．０， １Ｈ）， ７．３５（ｄ， Ｊ＝７．６Ｈｚ， ２Ｈ）， ７．１１（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．２３ （ｄ， １Ｈ）， ６．０１（ｓ， １Ｈ）， ５．５２（ｓ， １Ｈ）， ５．０６ （ｄ， Ｊ＝４．８Ｈｚ， １Ｈ）， ５．００－４．７０（ｍ， ２Ｈ）， ４ ４３（ｂｒｓ， １Ｈ）， ３．６０－３．５０（ｍ， １Ｈ）， １．８０－１．６０ （ｍ， ２０Ｈ）， １．５１ （ｓ， ３Ｈ），１．２０－１．１０（ｍ，１Ｈ）， １．０３（ｓ， ３Ｈ）， １．００－０．９５（ｍ， １Ｈ）。 6-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl) -2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][ Synthesis of 1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane-2-aminium 2,2,2-trifluoroacetate (INX-SM-38)

procedure:
In a 25 ml single neck round bottom flask, add (6-(4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-8b-(2-(( di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)carbamine Tert-butyl acid (0.17 g, 0.193 mmol) (INX-SM-38-2) was charged. TFA (0.17 mL) was added to this solution at 0° C., and the mixture was further stirred for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum. The crude product was subjected to preparative HPLC (column: New 11.7 min) to give the title compound as a white solid (0.025 g, 16.93%). LCMS [M ₊ H] ⁺ observed value for _C36H47NO9P was 668.4; ^1H NMR (400 MHz _, MeOD): δ: 7.47 (dd, J=10 & 2.0, 1H), 7.35 (d, J=7.6Hz, 2H), 7.11 (d, J=8.0Hz, 2H), 6.23 (d, 1H), 6.01 (s, 1H) , 5.52 (s, 1H), 5.06 (d, J=4.8Hz, 1H), 5.00-4.70 (m, 2H), 4 43 (brs, 1H), 3.60- 3.50 (m, 1H), 1.80-1.60 (m, 20H), 1.51 (s, 3H), 1.20-1.10 (m, 1H), 1.03 (s, 3H), 1.00-0.95 (m, 1H).

（２－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ，１０，１０－テトラメチル－４－オキソ－１，２，４，６ａ，６ｂ，７，８，８ａ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－８ｂＨ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－８ｂ－イル）－２－オキソエチル）リン酸ジ－ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－２１－１）の合成
手順：
１００ｍＬの一つ口丸底フラスコに、（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ，１０，１０－テトラメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（１．０ｇ、２．２１ｍｍｏｌ）、ジ－ｔｅｒｔ－ブチルＮ，Ｎ－ジイソプロピルホスホルアミダイト（１４．７ｇ、５３．０ｍｍｏｌ）、及びＤＭＦ（１ｍＬ）を充填した。この溶液に、１Ｈ－テトラゾール（１．５ｇ、２２．１ｍｍｏｌ）を添加し、室温で２４時間撹拌した。反応混合物を０℃に冷却し、過酸化水素（１０Ｖ）を添加し、室温で更に２時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ナトリウムチオサルフェート溶液に注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製の表題化合物を逆相カラムクロマトグラフィー（アセトニトリル／水）によって精製して、表題化合物（０．４ｇ、２８．０８％）を得た。Ｃ_３２Ｈ_４８Ｆ_２Ｏ_９Ｐに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、６４５．３［Ｍ＋Ｈ］^＋であった。 6-(4((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-( 2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[ Synthesis of 1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane-2-aminium 2,2,2-trifluoroacetate (INX-SM-21)

(2-((2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo-1 ,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1, 3] Synthesis procedure of di-tert-butyl dioxol-8b-yl)-2-oxoethyl)phosphate (INX-SM-21-1):
In a 100 mL one-neck round bottom flask, (2S, 6aS, 6bR, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a ,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5] Indeno[1,2-d][1,3]dioxol-4-one (1.0g, 2.21mmol), di-tert-butyl N,N-diisopropylphosphoramidite (14.7g, 53.0mmol) , and DMF (1 mL). To this solution was added 1H-tetrazole (1.5 g, 22.1 mmol) and stirred at room temperature for 24 hours. The reaction mixture was cooled to 0°C, hydrogen peroxide (10V) was added and stirred for a further 2 hours at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was poured into saturated sodium thiosulfate solution and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude title compound was purified by reverse phase column chromatography (acetonitrile/water) to give the title compound (0.4 g, 28.08%). LCMS [M+H] ⁺ observed value for C ₃₂ H ₄₈ F ₂ O ₉ P was 645.3 [M+H] ⁺ .

手順：
１００ｍＬの一つ口丸底フラスコに、ＤＣＭ（１０ｍＬ）中の（２－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ，１０，１０－テトラメチル－４－オキソ－１，２，４，６ａ，６ｂ，７，８，８ａ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－８ｂＨ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－８ｂ－イル）－２－オキソエチル）フォスフェート（ＩＮＸ－ＳＭ－２１－１）（０．２ｇ、０．３１ｍｍｏｌ）及びｔｅｒｔ－ブチル（６－（４－ホルミルベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ジ－ｔｅｒｔ－ブチル（０．１ｇ、０．３０ｍｍｏｌ）（ＩＮＸ－ＳＭ－３２－４）を充填した。ＭｇＳＯ_４（０．１８ｇ、１．５ｍｍｏｌ）及びＨＣｌＯ_４（０．１５ｇ、１．５ｍｍｏｌ）を０℃で添加し、室温で４時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を飽和ＮａＨＣＯ_３溶液に注ぎ、１０％ＭｅＯＨ：ＤＣＭで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、真空下で蒸発させた。粗製物を分取ＨＰＬＣ（カラム：Ｓｈｉｍ－Ｐａｃｋ Ｇｉｓｔ Ｃ１８、２０×２５０ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝７１：２９、保持時間１６．５分）により精製して、白色の固体として表題化合物を得た（０．０３３ｇ、１５．６％）。Ｃ_３６Ｈ_４５Ｆ_２ＮＯ_９Ｐに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、７０４．３であった；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）：δ：７．４０－７．３５（ｍ， ３Ｈ）， ７．１５（ｄ， Ｊ＝７．６Ｈｚ， ２Ｈ）， ６．３７－６．３４ （ｍ， ２Ｈ）， ５．６７－５．５２（ｍ， １Ｈ）， ５．５４（ｓ， １Ｈ， Ａｃｅｔａｌ－Ｈ）， ５．０７ （ｍ， Ｊ＝３．６Ｈｚ， １Ｈ， Ｃ１６－Ｈ）， ４．９５－４．７２（ｍ， ２Ｈ）， ４．３５－４．３０（ｍ， １Ｈ）， ３．７０－３．５８（ｍ， １Ｈ）， ２．７８－１．６３（ｍ， １９Ｈ）， １．６１ （ｓ， ３Ｈ）， １．０３ （ｓ， ３Ｈ）。 6-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b- (2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno Synthesis of [1,2-d][1,3]dioxol-10-yl)benzyl)spiro[3.3]heptane-2-aminium 2,2,2-trifluoroacetate (INX-SM-21)

procedure:
In a 100 mL single neck round bottom flask, add (2-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy- 6a,8a,10,10-tetramethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1' :4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl)phosphate (INX-SM-21-1) (0.2g, 0.31mmol) and Di-tert-butyl (6-(4-formylbenzyl)spiro[3.3]heptan-2-yl)carbamate (0.1 g, 0.30 mmol) (INX-SM-32-4) Filled. MgSO ₄ (0.18 g, 1.5 mmol) and HClO ₄ (0.15 g, 1.5 mmol) were added at 0° C. and stirred at room temperature for 4 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into saturated _NaHCO3 solution and extracted with 10% MeOH:DCM. The combined organic layers were dried over Na ₂ SO ₄ and evaporated under vacuum. The crude product was subjected to preparative HPLC (column: Shim-Pack Gist C18, 20 x 250 mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A:B = 71:29, retention time 16. 5 min) to give the title compound as a white solid (0.033 g, 15.6%). LCMS [M+H] ⁺ actual value for C ₃₆ H ₄₅ F ₂ NO ₉ P was 704.3; ¹ H NMR (400 MHz, MeOD): δ: 7.40-7.35 (m, 3H) , 7.15 (d, J=7.6Hz, 2H), 6.37-6.34 (m, 2H), 5.67-5.52 (m, 1H), 5.54 (s, 1H, Acetal-H), 5.07 (m, J=3.6Hz, 1H, C16-H), 4.95-4.72 (m, 2H), 4.35-4.30 (m, 1H), 3.70-3.58 (m, 1H), 2.78-1.63 (m, 19H), 1.61 (s, 3H), 1.03 (s, 3H).

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ－ジメチル－４－オキソ－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸（ＩＮＸ－Ａ－２３）（０．０５ｇ、０．０５３ｍｍｏｌ）及びＤＭＦ（１ｍＬ）を充填した。この溶液に、Ｌ－システイン（０．００９ｇ、０．０７４ｍｍｏｌ）を添加し、反応混合物を室温で２時間撹拌した。ＬＣＭＳによって示される反応の完了後、反応混合物を凍結乾燥し、粗製物を分取ＨＰＬＣ（カラム：ＮＥＷ Ｘｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ １９×２５０ｍｍ、５μｍ 移動相：Ａ＝水中の０．１％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝６７：３３、保持時間１１．０分）により精製して、白色の固体として表題化合物を得た（０．０５０ｇ、１５．９８％）。Ｃ_４８Ｈ_６１Ｆ_２Ｎ_４Ｏ_１３Ｓに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、９７１．６であった；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．３６－７．３４（ｍ， ３Ｈ）， ７．１６（ｄ， Ｊ＝８．０， ２Ｈ）， ６．３７－６．３２（ｍ， ２Ｈ）， ５．７０－５．４８（ｍ， １Ｈ）， ５．４８（ｓ， １Ｈ， アセタール－Ｈ）， ５．０８（ｄ， Ｊ＝４．０Ｈｚ， １Ｈ）， ４．６５（ｄ， １Ｈ）， ４．３７－３．３０（ｍ， ３Ｈ）， ４．１３－４．０９（ｍ， １Ｈ）， ４．０３－４．０１（ｍ， １Ｈ）， ３．９２（ｓ， ２Ｈ）， ３．３３（ｓ， ２Ｈ）， ３．３０－３．２７（ｍ， １Ｈ）， ３．１０－３．０５（ｍ， １Ｈ）， ２．８０－１．６４（ｍ， ２３Ｈ）， １．６０（ｓ， ３Ｈ）， ０．９２ （ｓ， ３Ｈ）。 (S)-4-(2-(2-(((R)-2-amino-2-carboxyethyl)thio)acetamido)acetamido)-5-((6-(4-((2S,6aS,6bR ,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a ,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole- Synthesis of 10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoic acid (INX A23-CYS)

procedure:
In a 10 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS) , 10R, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8 , 8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl) Spiro[3.3]heptan-2-yl)amino)-5-oxopentanoic acid (INX-A-23) (0.05 g, 0.053 mmol) and DMF (1 mL) were charged. To this solution was added L-cysteine (0.009 g, 0.074 mmol) and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction as indicated by LCMS, the reaction mixture was lyophilized and the crude was subjected to preparative HPLC (column: NEW Xbridge Prep, C18, OBD 19 x 250 mm, 5 μm mobile phase: A = 0.1% TFA in water, Purification by B=acetonitrile, A:B=67:33, retention time 11.0 min) gave the title compound as a white solid (0.050 g, 15.98%). LCMS [M+H] ⁺ observed value for C ₄₈ H ₆₁ F ₂ N ₄ O ₁₃ S was 971.6; ¹ H NMR (400 MHz, MeOD): δ: 7.36-7.34 (m, 3H), 7.16 (d, J=8.0, 2H), 6.37-6.32 (m, 2H), 5.70-5.48 (m, 1H), 5.48 (s, 1H, acetal-H), 5.08 (d, J=4.0Hz, 1H), 4.65 (d, 1H), 4.37-3.30 (m, 3H), 4.13-4. 09 (m, 1H), 4.03-4.01 (m, 1H), 3.92 (s, 2H), 3.33 (s, 2H), 3.30-3.27 (m, 1H) , 3.10-3.05 (m, 1H), 2.80-1.64 (m, 23H), 1.60 (s, 3H), 0.92 (s, 3H).

手順：
１０ｍＬの一つ口丸底フラスコに、（Ｓ）－４－（２－（２－ブロモアセトアミド）アセトアミド）－５－（（６－（４－（（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１０Ｒ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ－ジメチル－４－オキソ－８ｂ－（２－（ホスホノオキシ）アセチル）－２，４，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－１Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－１０－イル）ベンジル）スピロ［３．３］ヘプタン－２－イル）アミノ）－５－オキソペンタン酸（ＩＮＸ－Ａ－１２）（０．２ｇ、０．１９ｍｍｏｌ）及びＤＭＦ（１ｍＬ）を充填した。この溶液に、Ｌ－システイン（０．０３５ｇ、０．２９ｍｍｏｌ）を添加し、室温で１６時間撹拌した。ＬＣＭＳによって示される反応の完了後、反応混合物を凍結乾燥し、粗製物を分取ＨＰＬＣ（カラム：ｎｅｗ Ｘｂｒｉｄｇｅ Ｐｒｅｐ、Ｃ１８、ＯＢＤ １９×２５０ｍｍ、５μｍ 移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝７０：３０、保持時間１１．２分）により精製して、白色の固体として表題化合物を得た（０．０２５ｇ、１２．０２％）。Ｃ_４８Ｈ_６２Ｆ_２Ｎ_４Ｏ_１６ＰＳに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、１０５２．４であった；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．３８－７．３６（ｍ， ３Ｈ）， ７．１５（ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．３８－６．３４（ｍ， ２Ｈ）， ５．６８－５．５０（ｍ， １Ｈ， ＣＨ－Ｆ）， ５．５３（ｓ， １Ｈ， アセタール－Ｈ）， ５．０５（ｄ， Ｊ＝４．０Ｈｚ， １Ｈ）， ５．００－４．８３（ｍ， ３Ｈ）， ４．３４－４．３０（ｍ， ２Ｈ）， ４．２０－４．０８（ｍ， ２Ｈ）， ３．９１（ｓ， ２Ｈ）， ３．４３（ｓ， ２Ｈ）， ３．１１－３．０５（ｍ， １Ｈ）， ２．８０－１．７２（ｍ， ２３Ｈ）， １．６１（ｓ， ３Ｈ）， １．０３（ｓ， ３Ｈ） (S)-4-(2-(2-(((R)-2-amino-2-carboxyethyl)thio)acetamido)acetamido)-5-((6-(4-((2S,6aS,6bR ,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4 , 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3] Synthesis of dioxol-10-yl)benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoic acid (INX-A-12-CYS)

procedure:
In a 10 mL single-neck round bottom flask, (S)-4-(2-(2-bromoacetamido)acetamide)-5-((6-(4-((2S, 6aS, 6bR, 7S, 8aS, 8bS) ,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7 , 8,8a, 8b, 11a, 12,12a, 12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl) Benzyl)spiro[3.3]heptan-2-yl)amino)-5-oxopentanoic acid (INX-A-12) (0.2 g, 0.19 mmol) and DMF (1 mL) were charged. To this solution was added L-cysteine (0.035 g, 0.29 mmol) and stirred at room temperature for 16 hours. After completion of the reaction as indicated by LCMS, the reaction mixture was lyophilized and the crude was subjected to preparative HPLC (column: new Xbridge Prep, C18, OBD 19 x 250 mm, 5 μm mobile phase: A = 0.05% TFA in water, Purification by B=acetonitrile, A:B=70:30, retention time 11.2 min) gave the title compound as a white solid (0.025 g, 12.02%). LCMS [M+H] ⁺ observed value for C ₄₈ H ₆₂ F ₂ N ₄ O ₁₆ PS was 1052.4; ¹ H NMR (400 MHz, MeOD): δ: 7.38-7.36 (m, 3H), 7.15 (d, J=8.0Hz, 2H), 6.38-6.34 (m, 2H), 5.68-5.50 (m, 1H, CH-F), 5. 53 (s, 1H, acetal-H), 5.05 (d, J=4.0Hz, 1H), 5.00-4.83 (m, 3H), 4.34-4.30 (m, 2H) ), 4.20-4.08 (m, 2H), 3.91 (s, 2H), 3.43 (s, 2H), 3.11-3.05 (m, 1H), 2.80- 1.72 (m, 23H), 1.61 (s, 3H), 1.03 (s, 3H)

（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ，１０，１０－テトラメチル－４－オキソ－１，２，４，６ａ，６ｂ，７，８，８ａ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－８ｂＨ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－８ｂ－カルボン酸（ＩＮＸ－ＳＭ－１１－１）の合成

手順：
２５０ｍＬの丸底フラスコに、１，４－ジオキサン（８０ｍＬ）中の（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－８ｂ－（２－ヒドロキシアセチル）－６ａ，８ａ，１０，１０－テトラメチル－１，２，６ａ，６ｂ，７，８，８ａ，８ｂ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－４Ｈ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－４－オン（フルオシノロンアセトニド）（５．０ｇ、１１．０６ｍｍｏｌ）を充填した。この溶液に、Ｈ_２Ｏ（２０ｍＬ）中のＨＩＯ_４（３．２ｇ、３３．１８ｍｍｏｌ）を滴加し、室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をトリエチルアミンでクエンチし、減圧下で濃縮した。粗製物を更に２Ｍ ＮａＯＨ水溶液に注ぎ、酢酸エチルで洗浄した。水層を１Ｎ ＨＣｌで酸性化し、ＥｔＯＡｃで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、減圧下で濃縮して、黄色の固体として表題化合物を得た（４．０ｇ、８２．５６％）。Ｃ_２３Ｈ_２９Ｆ_２Ｏ_６に対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、４３９．２であった。 (2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)methyl)phenyl)-2, 6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2' Synthesis of ,1':4,5]indeno[1,2-d][1,3]dioxole-8b-carbothioic acid S-(cyanomethyl)2,2,2-trifluoroacetate (INX-SM-11)

(2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo-1,2,4 ,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole- Synthesis of 8b-carboxylic acid (INX-SM-11-1)

procedure:
In a 250 mL round bottom flask, (2S, 6aS, 6bR, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-2,6b-difluoro-7-hydroxy-8b-( 2-hydroxyacetyl)-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2', 1':4,5]indeno[1,2-d][1,3]dioxol-4-one (fluocinolone acetonide) (5.0 g, 11.06 mmol) was charged. To this solution was added _HIO4 (3.2 g, 33.18 mmol) in _H2O (20 mL) dropwise and stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with triethylamine and concentrated under reduced pressure. The crude was further poured into 2M aqueous NaOH and washed with ethyl acetate. The aqueous layer was acidified with 1N HCl and extracted with EtOAc. The combined organic layers were dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the title compound as a yellow solid (4.0 g, 82.56%). LCMS [M _{+ H} ] ⁺ observed value _{for C23H29F2O6} was 439.2.

手順：
１００ｍＬの丸底フラスコに、（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ，１０，１０－テトラメチル－４－オキソ－１，２，４，６ａ，６ｂ，７，８，８ａ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－８ｂＨ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－８ｂ－カルボン酸（ＩＮＸ－ＳＭ－１１－１）（４．０ｇ、９．１３ｍｍｏｌ）及びアセトン：Ｈ_２Ｏ（４０：１ｍＬ）を充填した。この溶液に、ジメチルカルバモチオ酸クロリド（２．２４ｇ、１８．２６ｍｍｏｌ）、ＴＥＡ（３．９ｍＬ、２７．３９ｍｍｏｌ）、及びＮａＩ（０．２７２ｇ、１．８２６ｍｍｏｌ）を室温で添加し、１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＤＭＡで希釈してそれを透明な溶液にし、次いで冷水に注いだ。得られた固体を濾過し、真空下で乾燥させて、黄色の固体として表題化合物を得、更に生成することなく次のステップに用いた（２．０ｇ、４１．７１％）。Ｃ_２６Ｈ_３４Ｆ_２ＮＯ_６Ｓに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、５２６．２であった。 (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo-1,2,4 ,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole- Synthesis of 8b-carboxylic acid dimethylcarbamic acid thioanhydride (INX-SM-11-2)

procedure:
In a 100 mL round bottom flask, (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4- Oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d] [1,3]Dioxole-8b-carboxylic acid (INX-SM-11-1) (4.0 g, 9.13 mmol) and acetone:H ₂ O (40:1 mL) were charged. Dimethylcarbamothioyl chloride (2.24 g, 18.26 mmol), TEA (3.9 mL, 27.39 mmol), and NaI (0.272 g, 1.826 mmol) were added to this solution at room temperature and stirred for 16 hours. did. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with DMA to make it a clear solution and then poured into cold water. The resulting solid was filtered and dried under vacuum to give the title compound as a yellow solid, which was used in the next step without further formation (2.0 g, 41.71%). LCMS _[ M ₊ H] ⁺ observed value _{for C26H34F2NO6S} was 526.2.

手順：
３５ｍＬのガラスバイアルに、（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ，１０，１０－テトラメチル－４－オキソ－１，２，４，６ａ，６ｂ，７，８，８ａ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－８ｂＨ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－８ｂ－カルボン酸ジメチルカルバミン酸チオ無水物（ＩＮＸ－ＳＭ－１１－２）（２．０ｇ、３．８０９ｍｍｏｌ）及びＤＭＡ（２０ｍＬ）を充填した。この溶液に、硫化水素ナトリウム（２．１３ｇ、３８．０９５ｍｍｏｌ）を添加し、室温で１時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を１Ｎ ＨＣｌでクエンチし、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、減圧下で濃縮して、黄色の固体として表題化合物を得た（０．５００ｇ、２８．９１％）。Ｃ_２３Ｈ_２９Ｆ_２Ｏ_５Ｓに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、４５５．２であった。 (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo-1,2,4 ,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole- Synthesis of 8b-carbothio S-acid (INX-SM-11-3)

procedure:
In a 35 mL glass vial, (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo -1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][ 1,3]dioxole-8b-carboxylic acid dimethylcarbamic acid thioanhydride (INX-SM-11-2) (2.0 g, 3.809 mmol) and DMA (20 mL) were charged. To this solution was added sodium hydrogen sulfide (2.13 g, 38.095 mmol) and stirred at room temperature for 1 hour. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with 1N HCl and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the title compound as a yellow solid (0.500 g, 28.91%). LCMS _[ M ₊ H] ⁺ observed value _{for C23H29F2O5S} was 455.2.

手順：
３５ｍＬのガラスバイアルに、（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ，１０，１０－テトラメチル－４－オキソ－１，２，４，６ａ，６ｂ，７，８，８ａ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－８ｂＨ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－８ｂ－カルボチオＳ－酸（ＩＮＸ－ＳＭ－１１－３）（０．５００ｇ、１．１０１ｍｍｏｌ）及び１，４－ジオキサン（５．０ｍＬ）を充填した。この溶液に、ＴＥＡ（０．３ｍＬ、２．２０ｍｍｏｌ）及びブロモアセトニトリル（０．１９７ｇ、１．６５１ｍｍｏｌ）を添加し、室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物をＨ_２Ｏに注ぎ、酢酸エチルで抽出した。組み合わせた有機層をＮａ_２ＳＯ_４上で乾燥させ、減圧下で濃縮した。粗生成物をカラムクロマトグラフィー（酢酸エチル：ヘキサン、３５：６５）によって精製して、褐色の固体として表題化合物を得た（０．１７ｇ、３１．３１％）。Ｃ_２５Ｈ_３０Ｆ_２ＮＯ_５Ｓに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、４９４．２であった。 (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo-1,2,4 ,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxole- Synthesis of 8b-carbothioic acid S-(cyanomethyl) (INX-SM-11-4)

procedure:
In a 35 mL glass vial, (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo -1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][ 1,3]dioxole-8b-carbothio S-acid (INX-SM-11-3) (0.500 g, 1.101 mmol) and 1,4-dioxane (5.0 mL) were charged. To this solution were added TEA (0.3 mL, 2.20 mmol) and bromoacetonitrile (0.197 g, 1.651 mmol) and stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was poured into _H2O and extracted with ethyl acetate. The combined organic layers were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate:hexane, 35:65) to give the title compound as a brown solid (0.17g, 31.31%). LCMS _[ M ₊ H] ⁺ observed value _{for C25H30F2NO5S} was 494.2.

手順：
３５ｍＬのガラスバイアルに、（２Ｓ，６ａＳ，６ｂＲ，７Ｓ，８ａＳ，８ｂＳ，１１ａＲ，１２ａＳ，１２ｂＳ）－２，６ｂ－ジフルオロ－７－ヒドロキシ－６ａ，８ａ，１０，１０－テトラメチル－４－オキソ－１，２，４，６ａ，６ｂ，７，８，８ａ，１１ａ，１２，１２ａ，１２ｂ－ドデカヒドロ－８ｂＨ－ナフト［２’，１’：４，５］インデノ［１，２－ｄ］［１，３］ジオキソール－８ｂ－カルボチオ酸Ｓ－（シアノメチル）（ＩＮＸ－ＳＭ－１１－４）（０．１５ｇ、０．３０ｍｍｏｌ）及びＤＣＭ（３ｍＬ）を充填した。この溶液に、（６－（４－ホルミルベンジル）スピロ［３．３］ヘプタン－２－イル）カルバミン酸ｔｅｒｔ－ブチル（ＩＮＸ－ＳＭ－３２－４）（０．１００ｇ、０．３０ｍｍｏｌ）、ＭｇＳＯ_４（０．１８３ｇ、１．５２ｍｍｏｌ）、及びＨＣｌＯ_４（０．１５２ｇ、１．５２０ｍｍｏｌ）を添加し、室温で１６時間撹拌した。ＴＬＣによって示される反応の完了後、反応混合物を真空下で蒸発させ、飽和ＮａＨＣＯ_３でクエンチした。固体を濾過し、粗製物を分取ＨＰＬＣ（カラム：ＳＨＩＭ－ＰＡＣＫ－ＧＩＳＴ Ｃ１８（２５０８（２５０×２０）ｍｍ、５μｍ、移動相：Ａ＝水中の０．０５％ＴＦＡ、Ｂ＝アセトニトリル、Ａ：Ｂ＝４９：５１、保持時間１６．５０分）により精製して、白色の固体として表題化合物を得た（０．０１０ｇ、４．３７％）。Ｃ_３７Ｈ_４３Ｆ_２Ｎ_２Ｏ_５Ｓに対するＬＣＭＳ［Ｍ＋Ｈ］^＋実測値は、６６５．５であった；^１Ｈ ＮＭＲ （４００ ＭＨｚ， ＭｅＯＤ）： δ：７．３８－７．３３（ｍ， ３Ｈ）， ７．１９ （ｄ， Ｊ＝８．０Ｈｚ， ２Ｈ）， ６．３７－６．３１（ｍ， ２Ｈ）， ５．６７－５．５０（ｍ， ２Ｈ）， ５．０１（ｄ， １Ｈ）， ４．３５－４．２５（ｍ， １Ｈ）， ３．９０（ｄｄ， Ｊ＝ １６．８Ｈｚ， ２Ｈ）， ３．６１－－３．５９（ｍ， １Ｈ）， ２．７０－１．６７ （ｍ， １９Ｈ）， １．５９（ｓ， ３Ｈ）， １．１２（ｓ， ３Ｈ）。 (2S, 6aS, 6bR, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((6-aminospiro[3.3]heptan-2-yl)methyl)phenyl)-2, 6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2' Synthesis of ,1':4,5]indeno[1,2-d][1,3]dioxole-8b-carbothioic acid S-(cyanomethyl)2,2,2-trifluoroacetate (INX-SM-11)

procedure:
In a 35 mL glass vial, (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-oxo -1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][ 1,3]dioxole-8b-carbothioic acid S-(cyanomethyl) (INX-SM-11-4) (0.15 g, 0.30 mmol) and DCM (3 mL) were charged. To this solution, tert-butyl (6-(4-formylbenzyl)spiro[3.3]heptan-2-yl)carbamate (INX-SM-32-4) (0.100 g, 0.30 mmol), MgSO ₄ (0.183 g, 1.52 mmol) and HClO ₄ (0.152 g, 1.520 mmol) were added and stirred at room temperature for 16 hours. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated under vacuum and quenched with saturated _NaHCO3 . The solid was filtered and the crude was purified by preparative HPLC (column: SHIM-PACK-GIST C18 (2508 (250 x 20) mm, 5 μm, mobile phase: A = 0.05% TFA in water, B = acetonitrile, A: B = 49:51, retention _time 16.50 min) to give the title compound as _a white solid (0.010 g, 4.37%) _{for C37H43F2N2O5S .} LCMS [M+H] ⁺ actual value was 665.5; ¹ H NMR (400 MHz, MeOD): δ: 7.38-7.33 (m, 3H), 7.19 (d, J=8 .0Hz, 2H), 6.37-6.31(m, 2H), 5.67-5.50(m, 2H), 5.01(d, 1H), 4.35-4.25(m , 1H), 3.90 (dd, J= 16.8Hz, 2H), 3.61--3.59 (m, 1H), 2.70-1.67 (m, 19H), 1.59 ( s, 3H), 1.12(s, 3H).

Example 4: Comparison of Binding and Internalization of Anti-VISTA Antibodies at Physiological pH To assess whether VISTA antibodies potentially effectively deliver steroids or other payloads to target immune cells, various A study was conducted to evaluate the internalization of anti-VISTA antibodies into human monocytes. Specifically, the binding and internalization of naked anti-human VISTA antibodies (INX200, 767 IgG1, 3 (antibody sequences in Figure 12), respectively) in human monocytes was compared. VISTA is highly expressed in most hematopoietic cells, especially bone marrow cells. Based on this, we speculated that rapid internalization combined with high density and selectivity for relevant cell types might make VISTA an ideal target for anti-inflammatory antibody drug conjugates (ADCs).

Drug conjugates with anti-EGFR antibodies have been extensively investigated because antibodies bound to EGFR are rapidly internalized by target cells. The literature details robust methods for determining internalization rates. For example, in EGFR-expressing cell lines, LA22 mAb against EGFR reached a maximum internalization level of 65.8% within 10 minutes at 37°C (Liu, Z., et al., (2009), “In-vitro internalization and in-vivo tumor uptake of anti-EGFR monoclonal antibody LA22 in A549 lung cancer cells and animal model ”, Cancer Biother Radiopharm, 15-20), whereas Ab033 showed 54% internalization in 15 minutes ( Durbin, K.R. et al., 2018, “Mechanistic Modeling of Antibody-Drug Conjugate Internalization at the Cellproton assignment r Level Reveals Inefficient Processing Steps”, Mol Cancer Ther, 1535-7163).

The purpose of this study was to develop a pH-sensitive anti-human VISTA with extended serum PK half-life developed by Five Prime Therapeutics and Bristol-Myers Squibb Company (Johnston, R.J. et al., (2019), “VISTA is to assess the internalization rate of anti-VISTA monoclonal antibody INX200 in human monocytes compared to 767-IgG1, an acidic pH-selective ligand for PSGL-1”, Nature, 574(7779), 565-570), and 767-IgG1. The purpose of this study was to further evaluate the internalization characteristics of , 3.

Materials and Methods In this experiment, the binding curve of anti-VISTA antibody INX200 and 767-IgG1,3 to human monocytes (from freshly isolated human peripheral blood mononuclear cells or PBMCS) was first determined. Second, the internalization rates of these different antibodies on human monocytes were defined using the non-internalizing antibody anti-CD45 as a negative control. Briefly, to detect only internalized antibodies, cells were incubated with fluorescently labeled antibodies for 30 minutes at 4°C, a temperature at which little or no internalization occurs. Cells were washed and incubated at 25°C for internalization. Equal amounts of anti-AF488 antibody were then used to quench cell surface signals at various time points. PBMCS were then stained with anti-CD14 antibody to identify monocytes and analyzed by flow cytometry.
Test Drug and Dosage - INX200 (Aragen, lot number BP-2875-019-6.1) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone that has L234A/L235A silencing mutations in the Fc region.
●Human IgG1si (BioXcell reference, lot number 659518N1) is an anti-RSV (respiratory syncytial virus) antibody having a human IgG1/kappa backbone with L234A/L235A silencing mutations in the Fc region.
●767-IgG1,3 (Aragen, lot number BP-2985-019-6) is a human IgG1,3 with L234A/L235E/G237A silencing mutations in the Fc region, developed by Five Prime Therapeutics and Bristol-Myers Squibb Company. gG1 /kappa backbone anti-human VISTA antibody. This antibody is designed to bind at low pH (e.g. pH 6) but minimally at physiological pH (pH 7.4) and is not subject to TMDD like other anti-VISTA antibodies. , resulting in a prolonged serum PK half-life. This antibody was produced as described in application W02018169993A1. The pH-sensitive behavior of the antibodies was confirmed via an ELISA format. Briefly, 767-IgG1,3 or INX200 was bound to the plate and hIX50-biotin (VISTA ECD) diluted in citrate/tween buffer containing BSA at pH 6.1, 6.7, or 7.5. and detected using streptavidin-HRP conjugate/TMB readout. Maximum INX200 binding was seen at pH 7.5, while minimum binding of 767-IgG1,3 was observed at pH 7.5, with increased levels of binding at pH 6.7 and a further increase at pH 6.1.
●CD45, clone HI30 is an anti-human CD45 monoclonal antibody.
- Anti-Alexa Fluor 488 (AF488) polyclonal antibody (Life Technologies, number A-11094) is an anti-Alexa Fluor 488 antibody used to quench the AF488 fluorescence signal.

All antibodies except anti-AF488 were conjugated to AF488 according to the manufacturer's instructions for labeling and purification (Invitrogen, catalog number A10235). Unless otherwise specified, antibodies were diluted in RPMI medium containing 1% BSA.

PBMC Preparation Human PBMC were isolated under sterile conditions from apheresis cones obtained from the Blood Donor Program at the Dartmouth Hitchcock Medical Center from healthy, unrelated human donors. Blood was transferred to a 50 ml Falcon tube and diluted to 30 ml with PBS. 13 ml of Histopaque 1077 (Sigma Aldrich) was slowly layered under the blood and the tube was centrifuged at 850 x g for 20 min at room temperature with gentle acceleration and no brake. Mononuclear cells were collected from the plasma/Ficoll interface, resuspended in 50 ml of PBS, and centrifuged at 300×g for 5 minutes. Cells were resuspended in PBS and then counted.

Fluorescent Labeling of Antibodies Anti-human VISTA antibodies and huIgG1si were conjugated with Alexa Fluor 488 dye according to the manufacturer's instructions for labeling and purification (Invitrogen, catalog number A10235). The concentration and extent of labeling was assessed by Nanodrop. The degree of labeling was 5.9 for INX200 and 7.1 for 767 IgG1,3. Anti-human CD45 (clone H130) conjugated with AF488 (Biolegend, no. 304017) and anti-CD14 against APC (clone M5E2, Biolegend, no. 301808) were used as received.

Analysis of antibody binding PBMC were resuspended at 5 x ¹⁰ cells/ml in RPMI/1% BSA buffer containing human Fc blocking reagent (eBioscience, 14-9161-73), then 50 μl/well of Cells were distributed into 96-well plates. Anti-human VISTA antibodies were prepared in a 2-fold dilution series (10 concentrations) starting at 333 nM (50 μg/ml) in RPMI/1% BSA buffer. PBMCS were stained on ice for 30 minutes to limit internalization, washed twice with PBS, and fixed with 2% FA in PBS for 10 minutes at 4°C. Monocytes were labeled with anti-CD14 mAb at 1:400 (v/v) in PBS/0.2% BSA for 20 minutes at room temperature. Cells were washed and analyzed by FACS using a Macsquant (Miltenyi) flow cytometer and FlowJo for analysis. All graphs were created with GraphPad (Prism).

Analysis of antibody internalization 5 × 10 ⁶ PBMC were resuspended in RPMI/1% BSA buffer containing 1 ml of human Fc blocking reagent (eBioscience, 14-9161-73) at 133 nM (20 μg/ml ) with anti-human VISTA mAb for 30 minutes on ice. Cells were washed with 3 ml of ice-cold PBS and centrifuged at 515×g for 2 minutes. PBMCS was resuspended in 1.25 ml fresh RPMI/1% BSA and kept at room temperature. By slowing internalization, a robust curve can be generated. At each time point, 50 μl of cells were transferred to a 96-well plate containing 50 μl of RPMI/1% BSA and anti-CD14 APC and total bound antibody was measured. Cells were kept on ice to block subsequent endogenous migration.

50 μl of cells were then incubated with 50 μl of RPMI/1% BSA containing anti-AF488 antibody at 266 nM (40 μg/ml) to quench the fluorescence of surface-bound antibodies, and anti-CD14 APC to label monocytes. The cells were transferred to a 96-well plate. Cells were kept on ice to block subsequent endogenous migration. Samples were collected in technical duplicate and antibody internalization was followed for up to 60 minutes. At the end of the time course, all samples were washed with PBS and fixed with 2% FA in PBS for 10 minutes at 4°C. After a final wash with PBS, cells were analyzed by FACS using a Macsquant (Miltenyi) flow cytometer and FlowJo for analysis. Median fluorescence intensity (MFI) of anti-VISTA or CD45 mAb was measured and data plotted.

Ｎ_１－各時点（ｔ１）でのクエンチされていないＭＦＩ
Ｏ_１－各時点（ｔ１）でのクエンチされたＭＦＩ
Ｎ_０－０分（ｔ０）でのクエンチされていないＭＦＩ
Ｑ_０－０分（ｔ０）での試料のクエンチされたＭＦＩ The subcellular fraction was calculated by subtracting the background fluorescence of untreated cells and normalizing the MFI value to MFI at time = 0. Internalization rate was calculated as the percentage of intracellular signal to total cell-associated fluorescence at each time point (see equation below) and normalized to 100% (Liao-Chan, S. et al., (2015) , “Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores”, PLoS O ne, 10(4): e012470).

N ₁ - unquenched MFI at each time point (t1)
O ₁ - quenched MFI at each time point (t1)
N _{0 −} Unquenched MFI at 0 min (t0)
Quenched MFI of the sample at Q ₀ −0 min (t0)

Binding of Naked Anti-VISTA Antibody (INX200, 767-IgG1,3) In the experiment of Figure 13, the median fluorescence intensity was measured for monocytes incubated with serial dilutions (0-333 nM) of the tested antibodies, and the black Dashed lines correspond to autofluorescence of unstained cells; n=1. A single measurement was made at each concentration. As shown there at physiological pH 7.3, INX200 showed a concentration-dependent increase in fluorescence within the range tested (0-333 nM) with CD14 ⁺ PBMCS (see Figure 13). In contrast, no signal was detected when cells were incubated with 767-IgG1,3 antibody. This was expected due to its selectivity for binding at lower pH, which was previously confirmed in an ELISA format. The human IgG1si antibody used to assess the level of non-specific binding showed little or no binding even at high antibody concentrations.

Internalization of anti-human VISTA antibodies Figure 14 shows the internalized fraction of anti-VISTA antibodies. In these experiments, the intracellular pool of cell-bound antibody was plotted over a 60 minute time course. For each data point, fluorescence was normalized to INX200 fluorescence at time 0 minutes. Mean±SD n=2 donors. To compare the internalized fraction of antibodies, MFI values at each time point were corrected for background fluorescence by subtracting the MFI of untreated monocytes and relative to the total MFI of INX200 at t=0 time point. (Figure 14). Consistent with the data shown in Figure 13, at t=0, 767-IgG1,3 showed 3.2% ± 4.5% weak binding compared to INX200 (Figure 14).

As shown there, the non-specific signal represented by human IgG1si staining was estimated at 5.9% at 0 minutes. When cells were incubated with INX200, a clear increase in intracellular signal was observed over time, with the intracellular fraction being 70.4% ± 9.2% by 60 min. MFI values reached a plateau by 40 minutes of time course. In contrast, only 5.3%±7.5% of 767-IgG1,3 was detected as an internal fraction at 60 minutes. The intracellular signal of human IgG1si was within 5-6% during the time course.

Additionally, in Figure 15, another experiment was performed to evaluate the internalization rate of INX200 antibody in monocytes over a 60 minute time course and compare it to the anti-CD45 antibody, HI30. As shown there, the anti-CD45 antibody was not internalized at any time point. Shown as mean±SD, n=2 donors. In contrast, INX200 was efficiently internalized, with half of the surface antibodies detected within cells by 20 minutes (Figure 15). Furthermore, within 40 minutes, 64.5%±11.2% of INX200 was internalized by monocytes. In contrast, no internalization of anti-CD45 mAb was observed at any time point tested.

The data show that anti-human VISTA INX200 binds with high affinity and is internalized by up to 64% internalization levels by 40 minutes. This makes VISTA uniquely suitable for delivering anti-inflammatory payloads to immune cells, as these results suggest that the majority of the payload should be delivered within a relatively short period of time. This strongly suggests a target, which is both non-trivial and important given the lack of CD45 internalization. In contrast, anti-human VISTA 767.3-IgG1.3, a pH-sensitive antibody, has limited binding to monocytes at physiological pH. Also, compared to INX200, 767-IgG1,3 showed negligible or limited levels of internalization at physiological pH.

Example 5: Comparison of PK of anti-VISTA antibodies as naked antibodies or dexamethasone conjugates Binding and internalization of exemplary anti-VISTA antibodies at physiological pH First experiment (experiment) in human VISTA knock-in (hVISTA KI) mice In 1), anti-human VISTA antibody INX200, naked or conjugated with dexamethasone (INX200A), and in the second experiment (experiment 2), 767-IgG1.3, naked or conjugated with dexamethasone (767-IgG1.3A). (Johnston et al, “VISTA is an acidic pH-selective ligand for PSGL-1.” Nature. 2019 Oct; 574(7779):565-5702019). Ta. These mice have human VISTA cDNA knocked in in place of the mouse VISTA gene and express human VISTA at both the RNA and protein levels. Experiments were performed in male hVISTA KI mice, and in both studies animals received one dose of antibody at 10 mg/Kg. The amount of antibody in peripheral blood was measured at 20 minutes, 4, 24, 48 hours, then on days 5, 8, 14, 21, and 28 for experiment 1, and on days 4, 7, 14, 21, and 28 for experiment 2. It was quantified visually.

The purpose of these two experiments was to confirm that the addition of eight linker payload molecules/antibodies corrects the PK, is a "pH sensitive" antibody as described by BMS/Five Prime Therapeutics, and that glucocorticoid binding Assess whether the forms have a significantly different PK (comparable to hIgG1) than anti-VISTA antibodies that bind to human VISTA-expressing cells in physiological and their respective glucocorticoid-bound forms (short compared to hIgG1) Was that.

Materials and Methods Experiment 1: PK study of INX200, INX200A (dexamethasone conjugate) in human VISTA KI mice hVISTA KI mice were divided into three groups of 10 each, and on day 0, 10 mg/Kg of human IgG1, INX200, and treated with INX200A.

Experiment 2: PK study of 767-IgG1.3, 767-IgG1.3A (dexamethasone conjugate) in human VISTA KI mice hVISTA KI mice were divided into three groups of 10 mice each, and each group was treated with 10 mg/Kg on day 0. Treated with human IgG1, 767-IgG1.3, and 767-IgG1A. In both experiments, mice were bled retroorbitally for 20 minutes, 4, 24, 48 hours and then on days 5 and 8 for experiment 1 and days 4 and 7 for experiment 2. Circulating antibodies were quantified by ELISA.
Test Drug and Dosage - INX200 (Aragen, Lot No. BP-2875-019-6.1) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone with L234A/L235A silencing mutations in the Fc region.
• INX200A (Abzena, lot number JZ-0556-005) is an INX200 antibody conjugated via an interchain disulfide with a drug/antibody ratio of 8. Linker/payload (A) consists of an esterase-sensitive linker and a dexamethasone payload.
●Human IgG1 (see BioXcell, lot number 659518N1)
●767-IgG1.3 (Aragen, lot number BP-2985-019-6) is a human IgG1.3 with L234A/L235E/G237A silencing mutations in the Fc region, developed by Five Prime Therapeutics and Bristol-Myers Squibb Company. gG1 / Kappa backbone anti-human VISTA antibody. This antibody is designed to bind at low pH, eg, pH 6, but minimally at physiological pH (pH 7.4) (1).
• 767-IgG1.3A (Abzena, lot number JCC0624003) is a 767-IgG1.3 antibody with a drug/antibody ratio of 8, conjugated via an interchain disulfide. Linker/payload (A) consists of an esterase-sensitive linker and a dexamethasone payload.

All antibodies were diluted in PBS and injected intravenously into the tail vein of mice in a volume of 0.2 ml to deliver a dose of 10 mg/Kg.

Mice hVISTA mice were bred at Sage Labs (Boyertown, PA). Mice, 8-12 weeks old, were initially transitioned for 3 weeks in an in-house quarantine facility and then transferred to a regular facility. They were allowed to acclimate for 1-2 weeks before starting the experiment.

Blood Sampling and Preparation Animals were bled no more than once every 24 hours. Each group of mice was divided into two subgroups of 5 mice and alternately bled on day 0. Blood was collected on day 0, 20 minutes, 4, 24, 48 hours post-injection, then on days 5 and 8 for experiment 1, and days 4 and 7 for experiment 2. In the first 24 hours, some data were excluded based on the registered quality of the intravenous injection. At subsequent time points, only animals that were successfully injected intravenously were bled.

Peripheral blood was collected from the retroorbital space using a glass Pasteur pipette that was first rinsed with heparin to prevent clotting. Blood was then centrifuged at 400 rcf for 5 minutes and plasma was collected and stored at -80°C for analysis (see above).

Antibody blood concentration analysis ELISA for detection of human IgG1
First, a 96-well flat bottom plate (Thermo Scientific Nunc Immunol Maxisorp, Cat. No. 442404) was incubated with 1 μg/ml mouse anti-huIgG Fcγ (Jackson ImmunoResearch, Cat. No. 209-005-098) in PBS at room temperature ( RT) for 1 hour Coated.

Wells were washed three times with PT (PBS with 0.05% Tween 20) and then blocked with PTB (PBS with 0.05% Tween 20 and 1% BSA) for 1 hour at room temperature. Human IgG (Southern Biotech, catalog number 0150-01) was used as a positive control and human IgG1 (BioXcell, catalog number BE0297) was used to construct a standard curve. Wells were washed three times with PT and then plasma samples were incubated in PTB at up to four different dilutions (to fit the standard curve) for 1 hour at room temperature.

After washing three times with PT, mouse anti-human IgG Fcγ conjugated with HRP (Jackson ImmunoResearch, catalog number 209-035-098) was used as the detection reagent at a dilution of 1/2000 and incubated for 1 hour at RT. After three washes, the ELISA reaction was revealed using TMB (Thermo Scientific, Cat. No. 34028) as a colorimetric substrate. After 5-10 minutes at room temperature, the reaction was stopped with 1M H ₂ SO ₄ .

ELISA for detection of INX200 or INX200A
First, a 96-well flat bottom plate (as described above) was coated with 1 μg/ml hIX50 (human VISTA ECD, made by Aragen Bioscience for ImmuNext) in PBS for 1 hour at RT. After washing three times, the wells were blocked with PTB for 1 hour at room temperature. INX908 (made by Aragen Bioscience for ImmuNext) was used as a positive control and a standard curve was constructed using INX200 or INX200A. Wells were washed three times with PT and then plasma samples were incubated in PTB at up to four different dilutions (to fit the standard curve) for 1 hour at room temperature.

After washing three times with PT, mouse anti-human kappa-HRP (Southern Biotech, Cat. No. 9230-05) was used as a detection reagent at 1/2000 and incubated for 1 hour at room temperature. After three washes, ELISA reactions were revealed using TMB substrate. After 5 minutes _at room temperature, the reaction was stopped with 1M _H2SO4 .

ELISA for detection of 767-IgG1.3 or 767-IgG1.3A
First, a 96-well flat bottom plate (as above) was coated with 1 μg/ml mouse anti-huIgG Fcγ (Jackson ImmunoResearch, Cat. No. 209-005-098) in PBS for 1 hour at room temperature.

After washing three times, the wells were blocked with PTB for 1 hour at room temperature. Human IgG (Southern Biotech, catalog number 0150-01) was used as a positive control and a standard curve was constructed using 767-IgG1.3 or 767-IgG1.3A. Wells were washed three times with PT and then plasma samples were incubated in PTB at up to four different dilutions (to fit the standard curve) for 1 hour at room temperature.

After washing three times with PTB, mouse anti-human IgG Fcγ-HRP (Jackson ImmunoResearch, catalog number 209-035-098) was used at 1/2000 as a detection reagent and incubated for 1 hour at room temperature. After three washes, ELISA reactions were revealed using TMB substrate according to the manufacturer's instructions. After 5 minutes _at room temperature, the reaction was stopped with 1M _H2SO4 . Antibody half-life was determined using the PKsolver program, which performs non-compartmental analysis (NCA) after an intravenous bolus.

Results Experiment 1: Plasma PK of naked or conjugated INX200 antibodies
Plasma samples from groups treated with INX200, INX200A, or hIgG1 were collected to determine antibody concentrations and their subsequent half-lives. INX200 exhibited a half-life of 0.1 day, which is low but consistent with previous PK data (T _1/2 = approximately 0.3 days), and the antibody fell below quantification levels in 24 hours. INX200A also showed the same PK. In contrast, human IgG1 had a half-life of 7.2 days, which is low but not atypical for immunoglobulins (Figure 16). The figure shows plasma concentrations of antibodies at annotated time points in hVISTA KI mice (SD; n=5/group).

Experiment 2: Plasma PK of naked or conjugated 767-IgG1.3 antibodies
Plasma samples from groups treated with 767-IgG1.3, 767-IgG1.3A, or hIgG1 were collected to determine antibody concentrations and their subsequent half-lives. The results showed that 767-IgG1.3 and 767-IgG1.3A exhibited similar half-lives of 3.5 and 4 days, respectively, and both were still detectable at day 7. The half-life of hIgG1 was 8.7 days, similar to that observed in Experiment 1 (see Figure 17, which includes PK studies for 767-IgG1.3, 767-IgG1.3A versus human IgG1). Plasma concentrations of antibodies at annotated time points (SD; n=5/group) in hVISTA KI mice.

Conclusion The results of these two experiments show that:

Experiment 1:
The data in Figure 16 show that the anti-human VISTA antibody INX200 (which binds to human VISTA cells at physiological pH) cannot be quantified in plasma 24 hours post-dose due to target-mediated pharmacokinetics (TMDD). However, the human IgG1 control shows an extended half-life more typical of IgG. The results further show that conjugation of dexamethasone with INX200 at DAR=8 does not affect its PK.

Experiment 2
The data in Figure 17 shows that the pH-sensitive anti-human VISTA 767-IgG1.3 exhibits similar PK to the human IgG1 control antibody, has limited binding to the VISTA target, and is not subject to TMDD. Also, conjugation of dexamethasone with 767-IgG1.3 at DAR=8 does not affect its PK.

Example 6: Long-term effects of antibody-drug conjugates on ex-vivo macrophage activation. Experiments were conducted to evaluate the long-term efficacy of exemplary antibody-drug conjugate (ADC) molecules of the present invention, including drugs and glucocorticoid (GC) drugs. We have previously shown that such ADCs exert robust anti-inflammatory activity in short-term inflammatory models (internal, unpublished work). The objectives of these studies were to (i) evaluate the pharmacodynamic range of various antibody drug conjugates (ADCs) and anti-human VISTA monoclonal antibodies conjugated with glucocorticoid (GC) payloads within bone marrow cells; (ii) To evaluate the efficacy of an exemplary INX GC linker payload ADC.

First, we compared the early GC response gene FKBP5 (Vermeer et al. (2003) “Glucocorticoid-induced increase in lymphocytic FKBP51 messen” with dexamethasone (Dex) on peritoneal resident macrophages (PRMs) and splenic monocytes. ger ribonucleic acid expression:a "potential marker for glucocorticoid sensitivity, potency, and bioavailability", J Clin Endocrinol Metab. 88(1):277-84). We evaluated the impact in Bo.

Based on this, we developed a model that allows assessing the long-term anti-inflammatory effects of ADCs on specific target populations such as PRM. Briefly, ADCs were delivered in vivo via intraperitoneal (i.p.) injection, and PRMs were isolated and cultured 1-7 days later. In the absence of GC treatment, PRM is highly activated after 2 hours as shown by increased cytokine production. In vivo Dex treatment 2 hours before PRM isolation significantly reduces cytokine production. The purpose of these studies was to evaluate the efficacy and pharmacodynamic range of the INX human VISTA antibody conjugated with a glucocorticoid payload compared to free Dex.

Materials and Methods Methods for assessing the effects of ADC or Dex on FKBP5 transcription in PRM and splenic monocytes Dex was injected intraperitoneally 2-24 hours before mice were euthanized and cells isolated. Mice were then injected with ADC 17 hours to 7 days before euthanasia and cell isolation.
Test drug and dosage Antibody INX201 (Aragen, lot number BP-3200-019-6) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone that has L234A/L235A/E269R/K322A silencing mutations in the Fc region. It is.
- INX201J (Abzena, lot numbers JZ-0556-025-1, JZ-0556-027, JZ-0556-013) is a 8.0 drug/ INX201 antibody with antibody ratio (DAR). The linker/payload (J) is based on a previously reported linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker and a budesonide analogue payload (INX J-2).
• INX231J (Abzena, lot number JZ-0556-013-1) is a conjugated INX231 with a DAR of 8.0. The linker/payload (INX J) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX J-2).
• INX234J (Abzena, lot number JZ-0556-013-2) is a conjugated INX234 with a DAR of 8.0. The linker/payload (INX J) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX J-2).
• INX240J (Abzena, lot number JZ-0556-013-3) is a conjugated INX240 with a DAR of 8.0. The linker/payload (INX J) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX J-2).
• INX201O (Abzena, lot number JZ-0556-016-2) is INX201 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX O) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-4).
• INX201P (Abzena, lot number JZ-0556-016-1) is INX201 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-3).
- INX233 (ATUM, lot number 82276.1.a) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone that has L234A/L235A/E269R/K322A silencing mutations in the Fc region.
• INX233P (Abzena, lot number PP-0924-001-3) is INX233 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
- INX231 (ATUM, lot number 72928.1.a) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone that has L234A/L235A/E269R/K322A silencing mutations in the Fc region.
• INX231P (Abzena, lot number JZ-0556-017-1) is INX231 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
- INX234 (ATUM, lot number 72931.2.a) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone that has L234A/L235A/E269R/K322A silencing mutations in the Fc region.
• INX234P (Abzena, lot number JZ-0556-017-2) is INX234 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-3).
- INX240 (ATUM, lot number 73419.2.a) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone that has L234A/L235A/E269R/K322A silencing mutations in the Fc region.
• INX240P (Abzena, lot number JZ-0556-017-3) is INX240 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-3).
• INX231R (Abzena, lot number PP-0924-001-2) is INX231 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX R) consists of a neutral protease-sensitive linker and a budesonide analog payload (INX-SM-3).
• INX231S (Abzena, lot number PP-0920-014-1) is INX231 with a DAR of 6.9, conjugated through complete modification of the interchain disulfide. The linker/payload (INX S) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-24).
• INX231V (Abzena, lot number PP-0920-014-2) is INX231 with a DAR of 7.8, conjugated through complete modification of the interchain disulfide. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX231W (Abzena, lot number PP-0920-014-3) is INX231 with a DAR of 7.5 conjugated through complete modification of the interchain disulfide. The linker/payload (INX W) consists of a positively charged protease-sensitive linker and a budesonide analog payload (INX-SM-3).
• INX234A3 (Abzena, lot number PP-0924-023-1) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (INX A3) consists of a positively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX234A4 (Abzena, lot number PP-0924-023-2) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 7.9. The linker/payload (INX A4) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-43).
• INX234T (Abzena, lot number PP-0924-023-3) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (INX T) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-3) and is phosphorylated.
• INX201L (Abzena, lot number JZ-0556-026-1) is an INX201 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (J) consists of a negatively charged protease-sensitive linker and a budesonide analogue payload (INX J-2) and is phosphorylated.
• INX234V (Abzena, lot number RJS-1054-003) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX234A5 (Abzena, lot number RJS-1054-002) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 7.9. The linker/payload (INX A5) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-44).
• INX234A11 (Abzena, lot number RJS-1054-001) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (INX A11) consists of a negatively charged protease-sensitive linker (Asn/gly) and a budesonide analog payload (INX-SM-32).
• INX231A7 (Abzena, lot number RJS-1054-007-001) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 7.8. The linker/payload (INX A7) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32) and is phosphorylated.
• INX231A12 (Abzena, lot number RJS-1054-007-002) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 6.99. The linker/payload (INX A12) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-25) and is phosphorylated.
• INX231A23 (Abzena, lot number RJS-1054-006-001) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 7.34. The linker/payload (INX A23) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-25).
- Antibodies were diluted in PBS and injected intraperitoneally (i.p.) in a volume of 0.2 ml to deliver the indicated doses.

Dexamethasone Dexamethasone Sterile Injection from Phoenix, NDC57319-519-05, was diluted in PBS and administered as described via intraperitoneal injection.

Mice hVISTA mice were bred at the Center for Comparative Medicine and Research at Dartmouth. All experiments were performed on female mice enrolled between 9 and 15 weeks of age.

Cell Isolation After being euthanized, mice were injected intraperitoneally with 7 ml of PBS/0.5% BSA/2mM EDTA. After briefly massaging the peritoneum, a small incision was made and peritoneal lavage fluid was collected. PRMs were isolated using negative selection (Miltenyi kit, reference 130-110-434). Spleens were dissected and mechanically dissociated, and monocytes were isolated using negative selection (Stem Cell, EasySep™ Mouse CD11b Positive Selection Kit II).

RNA preparation and real-time PCR
Cell pellets from different tissues were resuspended in 0.4 ml RNeasy lysis buffer from the RNeasy Plus mini kit (Qiagen, PN: 74136) and homogenized 5 times with a 20G needle. RNA was isolated according to the manufacturer's instructions and RNA was eluted in 30 or 40 ml H ₂ O (RNase/DNase free). RNA concentration was assessed on a Nanodrop.

Reverse transcription was performed using Taqman reverse transcription reagents (number N8080234) according to the manufacturer's instructions. Quantitative real-time PCR was performed using the Taqman Master Mix 2X kit (number 4369016) and Taqman primers for mouse FKBP5 (Mm00487401_m1) and mouse HPRT (Mm446968_m1) as the housekeeping gene, using QuantStud from Applied Biosystems. Run on io3 did.

Ct data were converted to DCt (FKBP5 normalized to HPRT within the sample) and then to ΔΔCt (relative FKBP5 levels of treated samples vs. PBS control) to obtain Log2 fold change versus PBS.

Peritoneal resident macrophage culture and cytokine analysis Culture conditions PRMs were resuspended in RPMI 1640 containing 10% FBS, 10 mM Hepes, penicillin/streptomycin, and glutamine at 100,000 cells per well in a 96-well tissue culture plate. was plated. Supernatants were collected 2 and 24 hours after plating and stored at -80°C.

Cytokine analysis using the Millipore platform Cytokine analysis was performed on 25 ml of plasma using the Millipore mouse 32-plex platform. The Immune Monitoring Laboratory (IML, a shared resource of the Dartmouth-Hitchcock Norris Cotton Cancer Center) performed the analysis.
Cytokine analysis via ELISA ●BioLegend (Cat. No. 430904) ELISA MAX Deluxe Set Mouse TNF-α
●BioLegend (Cat. No. 431304) ELISA MAX Deluxe Set Mouse IL-6

ELISA was performed according to the manufacturer's attached protocol.

Results Experiment 1: FKBP5 transcriptional activation after Dex treatment in peritoneal resident macrophages and splenic monocytes. Compare the effects of FKBP5 transcriptional activation. As shown therein, the Dex (left) effect was evaluated at 4 and 24 hours after a single intraperitoneal injection at 2 mg/Kg, and the ADC (right) effect with a GC payload of 0.2 mg/Kg. Analyzed at 24, 48, 72 and 96 hours after a single intraperitoneal injection at 10 mg/Kg delivered. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus PBS control group. Four mice per group were pooled together to generate sufficient material for RNA preparation. The data in Figure 18 show that Dex treatment causes a dramatic increase in FKBP5 messenger RNA in both PRM and splenic monocytes by 4 hours post-treatment, but the effects on transcription disappear by 24 hours. (Figure 18, left). In contrast, the effect of INX201J on FKBP5 is long-term, i.e., increases in FKBP5 messenger RNA are detected as late as 96 hours in PRM and 72 hours in splenic monocytes (FIG. 18, right).

In the absence of additional stimulation, when PRMs are transferred to tissue culture plates, they are activated very rapidly, significantly increasing the production of numerous pro-inflammatory cytokines that can be measured from cell supernatants as early as 1 hour after seeding. increase to

Experiment 2: Dex treatment prevents ex vivo induction of proinflammatory cytokines in PRMs The experiment in Figure 19 shows that Dex treatment prevents ex vivo induction of proinflammatory cytokines in PRMs. In the experiment, Dex effects were evaluated 2 hours after a single intraperitoneal injection at 2 mg/Kg and tested in mice 32-plex (see Methods section) (n = 4 mice/group; unpaired T-test. ) was used to assess IL-6 and TNFα in cell supernatants (collected at 1 hour).

These results demonstrate that in vivo Dex treatment 2 hours before cell isolation robustly shuts down ex vivo PRM activation, exemplified by IL-6 and TNFα secretion, as early as 1 hour after cell seeding. Show what you can do. This effect is still clearly detected even after 24 hours of culture (not shown).

Experiment 3: Pharmacodynamics of ADC INX201J In the experiment included in Figure 20, the pharmacodynamic range of ADC INX201J was evaluated by injecting the ADC -4, -2, and -1 days before PRM isolation and ex vivo stimulation. did. Dex control group was injected 2 hours before PRM isolation. Therein, we evaluated Dex effects 2 hours after a single intraperitoneal injection at 2 and 0.2 mg/Kg, and 1 day after an injection at 10 mg/Kg (equivalent to a payload of 0.2 mg/Kg). The INX201J effect was evaluated on days (d-1), 2 days (d-2), and 4 days (d-4). Cell supernatants were collected at 2 hours. TNFα was measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group). INX201J was administered in an amount corresponding to a payload of 10 mg/Kg or 0.2 mg/Kg.

As shown in Figure 20, INX201J treatment had a highly significant effect on reducing TNFα production when administered on day −1. In addition, administration of ADC on day −2 or −4 instead affected secreted cytokine levels similarly to the Dex control group. Data suggest that INX201J causes long-term (>4 days) anti-inflammatory effects on PRM. Notably, the amount of TNFα detected in the supernatant in this experiment is much lower than in the previous experiment, probably due to the fact that quantification was performed by ELISA (instead of Luminex). We also observed lower IL-6 levels when quantification was performed by ELISA in other experiments.

Experiment 3: Long-term efficacy of different anti-VISTA antibodies conjugated to J payload In the experiment of Figure 21, we evaluated the long-term efficacy of different anti-VISTA antibodies conjugated to J payload. INX201J, INX234J, and INX240J were injected at 10 mg/Kg (0.2 mg/Kg payload) on days -4 and -7, and Dex was administered at 2 mg/Kg 2 hours before cell isolation. . For practical experimental reasons, INX231J was administered only on day -4.

The results in Figure 21 revealed that the ADCs tested had a long-term effect on the ex vivo induction of TNFα and IL-6 in PRM. Among them, Dex effects were evaluated 2 hours after a single intraperitoneal injection at 2 mg/Kg, and 4 days (-4) and 7 days (-7) after a single intraperitoneal injection at 10 mg/Kg. The effects of INX201J, INX231J, INX234J, and INX240J were evaluated. Cell supernatants were collected at 2 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group). As shown in Figure 21, all four ADCs exhibited potent anti-inflammatory activity for both TNFα and IL-6 when administered on either day -4 or day -7.

Experiment 5: Dose-dependent effects of INX231J, INX234J, and INX240J on ex-vivo PRM activation In the experiment of Figure 22, the dose-dependent effects of INX231J, INX234J, and INX240J on ex-vivo PRM activation were evaluated. In these experiments, Dex effects were evaluated 2 hours after a single intraperitoneal injection at 2 mg/Kg and INX231J, INX234J, and INX240J efficacy was evaluated 7 days after a single intraperitoneal injection with GC payload (equivalent to GC payload). Cell supernatants were collected at 2 hours. TNFα and IL-6 was measured.

As described, all ADCs were injected intraperitoneally on day −7 at different doses: 10, 3, and 1 mg/Kg with GC payloads of 0.2, 0.06, and 0.02 mg/Kg, respectively. Delivered. Dex was administered at 2 mg/Kg 2 hours before cell isolation. The results showed that no significant differences were observed between the different ADCs, suggesting that they had similar efficacy (Figure 22).

Experiment 6: Efficacy of an exemplary ADC of the present invention compared to Dex 7 days after treatment and INX201 conjugated with linker/payload P. In the experiment of Figure 23, 1) compared to Dex 7 days after treatment; 2) the efficacy of INX201 conjugated with linker/payload P was evaluated. The results in Figure 23 show that INX201J, INX201P, INX231J, INX234J, and INX240J ADCs are equally effective in preventing ex vivo induction of TNFα and IL-6 during PRM. The experiment evaluated the effects of INX201J, INX201P, INX231J, INX234J, INX240J, and Dex 7 days after a single intraperitoneal injection, with ADC at 10 mg/Kg (0.2 mg/Kg GC payload) and Dex at 2 mg. /Kg. Cell supernatants were collected at 2 hours. TNFα and IL-6 was measured.

All treatments were performed by intraperitoneal injection on day −7, with a payload of 2 mg/Kg for Dex and 0.2 mg/Kg for ADC, as described above. The data in Figure 23 showed that Dex lost all efficacy in controlling cytokine responses, while all ADCs carrying J or P payloads had comparable efficacy.

Experiment 7: Efficacy of an exemplary ADC of the present invention compared to INX201 conjugated with Dex and linker/payload P 7 days after treatment In this experiment of FIG. 24, different anti-VISTA antibodies compared to INX201J The impact of altering the anti-VISTA CDRs on extended efficacy was evaluated by evaluating the INX P payload conjugated with VISTA. All ADCs were injected intraperitoneally on day -7 at a dose of 0.2 mg/Kg payload.

The data showed that all anti-VISTA ADCs carrying INX J or INX P payloads had comparable potency after extended periods of time (Figure 24). Figure 24 shows that INX201J, INX231P, INX234P, and INX240P ADCs are equally effective in preventing ex vivo induction of TNFα during PRM. ADC efficacy was evaluated 7 days after a single intraperitoneal injection, and ADC was administered at 10 mg/Kg (GC payload of 0.2 mg/Kg). Cell supernatants were collected at 2 hours. TNFα was measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM).

Experiment 8: Long-term efficacy of INX R linker payload conjugated with INX231.
In this experiment of Figure 25, the long-term efficacy of INX R linker payload conjugated with INX231 was evaluated. This conjugate is similar to INX231P, but contains a neutral dipeptide linker, whereas INX231P has a negatively charged dipeptide linker. The efficacy of an ADC containing the additional anti-VISTA antibody INX233 was also evaluated. INX231P and INX234P were used as comparison controls. All ADCs were injected intraperitoneally on day -7 at a dose of 0.2 mg/Kg payload. Cell supernatants collected at 24 hours were analyzed as in the previous experiment. No significant differences were observed between supernatants collected at 2 or 24 hours.

The data further showed that INX231R and INX233P had comparable potency to INX234P (Figure 25), with the exception of INX231P, which showed lower potency than normal (see experiment 6). In particular, the data in Figure 25 show that INX231P, INX231R, INX233P, and INX234P are equally effective in preventing ex vivo induction of TNFα and IL-6 during PRM. In the experiment, ADC efficacy was evaluated 7 days after a single intraperitoneal injection, and ADC was administered at 10 mg/Kg (GC payload of 0.2 mg/Kg). Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM).

Experiment 9: Long-term efficacy of INX R linker payload conjugated with INX231.
In this experiment of Figure 26, the long-term efficacy of several other INX linker payloads conjugated with INX231 was evaluated. In this experiment, we independently varied the charge of the dipeptide linker (INX R, INX W vs. INX P), the halogenation of the steroid ring (INX S vs. INX P), and the payload (INX V vs. INX P). A linker payload INX O with a different payload than INX P was also evaluated as an INX201 conjugate. All ADCs were injected intraperitoneally on day -7 at a dose of 0.2 mg/Kg payload. Cell supernatants collected at 24 hours were analyzed.

As shown from the data in Figure 26, linker payloads INX S, INX V, and INX W conjugated with INX231 showed significant long-term efficacy in controlling cytokine responses, whereas INX231P and INX231R Showing more limited but significant long-term efficacy, the INX O payload conjugated with INX231 had little and non-significant effect on PRM cytokine responses, and Figure 26 shows that TNFα and IL- Figure 2 shows an evaluation of the efficacy of GC linker payloads INX R, INX O, INX S, INX V and INX W versus INX P conjugated with INX231 in preventing ex vivo induction of 6. ADC efficacy was evaluated 7 days after a single intraperitoneal injection, and ADC was administered at a GC payload of 0.2 mg/Kg. Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM).

Experiment 10: Efficacy of payloads INX S, INX V, and INX W in the pharmacodynamic range at 1, 7, and 14 days.
The present experiment in Figure 27 evaluated the long-term efficacy of payloads INX S, INX V, and INX W, focusing on the pharmacodynamic range at 1, 7, and 14 days. INX231P, INX231S, and INX231V were injected intraperitoneally on days -14, -7, or -1, and INX231W was injected only on day -14. All ADCs were administered at a dose of 0.2 mg/Kg payload. PRMs were collected on day 0. A portion of the cells was used for RNA isolation and the remaining cells were cultured. Cell supernatants collected 24 hours after isolation were analyzed.

As shown from the data in Figure 27, after 24 hours (day 1/D1) or 7 days, payloads INX S and INX V show similar levels of induction of FKBP5 as INX P. After 14 days, there is an obvious decrease in FKBP5 transcription in the group treated with INX P, while groups treated with payloads INX S, V, and W still show high levels of FKBP5 transcription. Figure 27 contains results of efficacy evaluation of GC payloads INX231S, INX231V, and INX231W versus INX231P in inducing FKBP5 transcription in PRM. ADC efficacy was evaluated at 1, 7, and 14 days after a single intraperitoneal injection, and ADC was administered at a GC payload of 0.2 mg/Kg. FKBP5 expression was measured by quantitative real-time PCR and presented as Log2 fold change versus PBS control group (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM).

The experiment in Figure 28 further shows that 24 hours after treatment, payloads INX P, INX S, and INX V have similar efficacy in preventing TNFα and IL-6 production by macrophages. Dosing 7 days earlier showed similar data, with INX231S being slightly more potent. Administration before 14 days resulted in loss of functional activity of INX231P, INX231V, and INX231W, whereas INX231S retained some activity (reduction in IL-6 on day 14 was significantly different p=0.0669 ). The data in FIG. 28 specifically shows the efficacy evaluation of GC payloads INX231S, INX231V, and INX231W versus INX231P in preventing ex vivo induction of TNFα and IL-6 in PRM. ADC efficacy was evaluated at 1, 7, and 14 days after a single intraperitoneal injection, and ADC was administered at a GC payload of 0.2 mg/Kg. Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM).

Experiment 11: Efficacy of Conjugates INX234A3, INX234A4, INX234T, INX201L, and INX231S Focused on Days 1, 7, and 14. , and INX231S were evaluated, focusing on the pharmacodynamic range at 1, 7, and 14 days. All ADCs were injected intraperitoneally on days -14, -7, or -1 at a dose of 0.2 mg/Kg payload. PRMs were collected on day 0. A portion of the cells was used for RNA isolation and the remaining cells were cultured. Cell supernatants collected 24 hours after isolation were analyzed. Isolated splenocytes were also collected and used to analyze FKBP5 expression levels.

The data in Figure 29 show that all payloads tested induced similarly high levels of FKBP5 transcription at 24 hours. After 7 days, there was a general decrease in FKBP5 transcription in all groups. By day 14, only the INX201L treatment group showed a continued decrease in FKBP5, whereas INX234A3, INX234A4, INX234T, and INX231S appeared to induce stable FKBP5 transcript levels unchanged from day 7 onwards. be.

In previous experiments, almost no FKBP5 signal was observed in the spleen when INX J and INX P payload conjugates were analyzed more than 4 days after injection. Very interestingly, the experiment in Figure 29 testing the efficacy of INX234A4, INX234T, INX201L, and INX231S showed significant induction of FKBP5 at day 7, and further by day 14, both INX234T and INX231S still induced a significant increase. More specifically, Figure 29 shows efficacy evaluation of GC payloads INX234A3, INX234A4, INX234T, INX201L and INX231S in inducing FKBP5 transcription in peritoneal resident macrophages (top row) and splenocytes (bottom row). ADC efficacy was evaluated at 1, 7, and 14 days after a single intraperitoneal injection, and ADC was administered at a GC payload of 0.2 mg/Kg. FKBP5 expression was measured by quantitative real-time PCR and presented as Log2 fold change versus PBS control group (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM).

The experiment in Figure 30 further shows that 24 hours after treatment, all payloads INX A3, INX A4, INX T, INX L, and INX S had similar efficacy in preventing TNFα and IL-6 production by macrophages. Indicates that it has. Administration 7 days earlier showed similar results to INX234A3, with INX231S being slightly more potent. Administration 14 days earlier resulted in loss of functional activity of INX234A4, which controls both TNFα and IL-6 production, but interestingly, INX234A3, INX234T, INX201L, and INX231S still significantly inhibited TNFα production. decreased. More specifically, the experiments in Figure 30 evaluate the efficacy of GC payloads INX234A3, INX234A4, INX234T, INX201L, and INX231S in preventing ex vivo induction of TNFα and IL-6 in PRM. These ADC effects were evaluated at 1, 7, and 14 days after a single intraperitoneal injection, and ADCs were administered at a GC payload of 0.2 mg/Kg. Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM).

Experiment 12: Efficacy of conjugates INX234A5 and INX234A11 vs. INX234V at 7 and 14 days In this experiment in Figure 31, we investigated the efficacy of two other payloads in macrophage assays, namely INX234A5 and INX234A11 vs. INX234V at 7 and 14 days. The evaluation focused on the pharmacodynamic range of All ADCs were injected intraperitoneally on day 0 at a dose of 0.2 mg/Kg payload. PRMs were collected on days 7 and 14. A portion of the cells was used for RNA isolation and the remaining cells were cultured. Cell supernatants collected 24 hours after isolation were evaluated. As shown by the data in Figure 31, all payloads induced similarly high levels of FKBP5 transcription 7 days post-injection. By day 14, there is a general decrease in FKBP5 transcription, but there is still a significant difference compared to the control group.

More specifically, the experiments in Figure 31 assayed the efficacy of GC payload INX234V, INX234A5 in inducing FKBP5 transcription in peritoneal resident macrophages and assessed ADC effects at 7 and 14 days after a single i.p. injection. These ADCs were administered with a GC payload of 0.2 mg/Kg. FKBP5 expression was measured by quantitative real-time PCR and presented as Log2 fold change versus PBS control group (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM).

The experiment in Figure 32 further shows that after 14 days, all tested ADCs retain similar efficacy in preventing TNFα and IL-6 production by macrophages. In particular, the experiment in Figure 32 compares the efficacy of GC payloads INX234V, INX234A5, and INX234A11 in preventing ex vivo induction of TNFα and IL-6 in PRM. The efficacy of these ADCs was evaluated 14 days after a single intraperitoneal injection, and the ADCs were administered at a GC payload of 0.2 mg/Kg. Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM).

Experiment 13: Efficacy of Conjugates INX231A7, INX231A12, and INX231A23 vs. INX234V at Days 7, 14, and 21 In this experiment in Figure 33, three other payloads in the macrophage assay, namely, INX231A7, INX231A12, and INX231A23 vs. INX234V Efficacy was evaluated focusing on the pharmacodynamic range at 7 and 21 days post-injection. All ADCs were injected intraperitoneally on day 0 at a dose of 0.2 mg/Kg payload, except INX231A7, which was administered at a payload of 0.08 mg/Kg. PRMs were collected on day 7. A portion of the cells was used for RNA isolation and the remaining cells were cultured. Cell supernatants collected 24 hours after isolation were evaluated. More specifically, these experiments in Figure 33 evaluated the efficacy of GC payloads INX234V, INX231A7, INX231A12, and INX231A23 in inducing FKBP5 transcription in peritoneal resident macrophages, and showed that 7 and 7 after a single i.p. ADC efficacy was evaluated at day 21, and ADCs were administered at a GC payload of 0.2 mg/Kg, except for INX231A7, which was administered at a payload of 0.08 mg/Kg. FKBP5 expression was measured by quantitative real-time PCR and presented as Log2 fold change versus PBS control group (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM).

As shown by the data in Figure 33, all tested payloads induced similarly high levels of FKBP5 transcription 7 days post-injection, with INX231A23 being slightly more potent. By day 21 post-injection, a small but significant increase in FKBP5 transcription was still detected compared to the PBS-treated group, except for the INX231A7-treated group.

The results of the experiments contained in Figure 34 further demonstrate that after 7, 14, and 21 days, all tested ADCs retain similar efficacy in preventing TNFα and IL-6 production by macrophages. and here again it is further shown that INX231A23 is the most effective, especially with respect to TNFa production. At 14 days post-injection, a significant decrease in TNFa was observed in all ADCs tested, and by 21 days post-dose, there was still a significant decrease in TNFa production in the INX231A7, INX231A12, and INX231A23-treated groups. Ta.

More specifically, FIG. 34 shows the results of efficacy evaluation of GC payloads INX234V, INX231A7, INX231A12, and INX231A23 in preventing ex vivo induction of TNFα and IL-6 in PRM. In these experiments, ADC efficacy was evaluated at 7, 14, and 21 days after a single intraperitoneal injection, and ADC was administered at 0.2 mg/Kg GC payload. Cell supernatants were collected at 24 hours. TNFα and IL-6 were measured using ELISA (see Methods section) (n=4 mice/group; conventional one-way ANOVA compared to PBS only group, SEM). Of particular note is the fact that even though INX231A7 was administered at only 0.08 mg/Kg payload, it still had a significant effect on TNFa production after 21 days.

Conclusion The data show that:
A single injection of INX201J induces long-term transcriptional induction of the GC reporter transcript FKBP5 in PRM target cell populations, demonstrating that the pharmacodynamic range of ADC extends beyond 4 days, whereas FKBP5 transcriptional induction by Dex Undetectable by 24 hours.
- Linker payloads INX S, INX V, INX W, INX A3, INX A4, INX A5, INX A11, and INX T still exhibit significant efficacy when administered 14 days prior to FKBP5 induction.
- Significant FKBP5 induction was observed on days 1, 7, and 14 in splenocytes containing analogs of INX P, specifically INX T (phosphorylated) and INX S (fluorinated at C6/C9). It was done. INX A4, and the previous document, INX L (phosphorylated version of INX J), show significant induction only on days 1 and 7.

In a model for in vivo treatment/ex vivo evaluation of PRM inflammatory conditions based on pro-inflammatory IL-6 and TNFα production/secretion:
- The tested ADCs (INX231J/INX234J/INX240J) show efficacy after 7 days even when administered with a payload of 0.02 mg/Kg, which is 100 times less than the Dex control administered at 2 mg/Kg.
- All ADCs tested (INX201J/INX201P/INX231J/INX234J/INX240J) have a pharmacological range of PRM based on reduction of TNFα and IL-6 of 7 days or more, whereas Dex loses all efficacy. There is.
- Linker/payloads INX R, INX P, and INX J show similar efficacy when administered 7 days earlier.
- Linker/payloads INX S, INX V, and INX W appear significantly more potent than payloads INX P and INX R when administered 7 days earlier. For INX V and INX W, this may be due to an expansion release. In the case of INX S, this may be due to the potency of the C6/C9 fluorinated free payload.
- Linker payload INX O shows very limited and non-significant potency when conjugated with INX201.
- Linkers/payloads INX S, INX V, INX A3, INX T, INX L, INX A5, and INX A11 had significant efficacy in controlling cytokine responses in PRM when administered 14 days prior.
- Linkers/payloads INX A7, INX A23, and INX A12 induce efficacy at least 21 days after injection.

Of particular note in these assays, all analogs of INX V, including spiro[3.3]heptane, remain potent for at least 7 days after injection, based on their ability to induce FKBP5 and cytokine reduction in an ex vivo PRM model. That is what happened. Analogs tested that were shown to be effective for at least 7 days include INX A3, INX A4, INX A5, INX A7, INX A11, INX A12, and INX A23, and further include:
- Phosphorylated analog (INX A12/INX A7)
- Double fluorinated analogs (INX A23/A12)
- Ether variants of methylene between the phenyl ring and spiro[3.3]heptane (INX A4 and INX A5)
- Linker variants of negatively charged gly/glu linkers, including positively charged lys/gly (INX A3), negatively charged Asn/gly (INX A11)

Example 7: Effect of Antibody Drug Conjugates on LPS-Induced Inflammation To evaluate the effect of exemplary antibody-drug conjugates according to the invention on LPS-induced inflammation, results are disclosed in this example and shown in Figures 35-48. Fourteen in vivo studies were performed as indicated.

A short-term model of LPS-induced systemic inflammation was used to evaluate the potential efficacy of ADCs in autoimmune diseases. Intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) is widely used as a model of both local and systemic acute immune responses in mice. The LPS model is characterized by a burst of pro-inflammatory cytokines in the blood circulation, which can be monitored as early as 2 hours after injection. By 24 hours, most cytokines return to normal levels. This model was utilized primarily by monitoring cytokine responses 2 or 4 hours after LPS injection. Preliminary studies have shown that dexamethasone (Dex) treatment has a dose-dependent effect on IL-12p40, TNFα, MIG, MIP-1α, and IL-1β, detectable as early as 2 hours. Therefore, studies focused on measuring one or more of these five cytokines. (Vermeer et al. (2003) Glucocorticoid-induced increase in lymphocytic FKBP51 messenger ribonucleic acid expression: a potential marker for glucocorticoid sensitivity, potency, and bioavailability. J Clin Endocrinol Metab. Jan; 88(1):277-84) .

The purpose of the study was to evaluate the efficacy of human anti-VISTA antibodies conjugated with various glucocorticoid payloads compared to free Dex.

Materials and Methods Methods In these experiments, mice received antibody or Dex treatment approximately 20 hours or 2-4 hours prior to LPS injection, respectively. Dex is short lived and acts quickly, whereas ADC requires additional processing time. These time points were chosen as a way to fairly compare the peak activities of ADC and Dex.

Blood was collected 2 or 4 hours after intraperitoneal injection of LPS and plasma isolated for cytokine analysis.
Test drug and dosage Antibody INX201 (Aragen, lot number BP-3200-019-6) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone that has L234A/L235A/E269R/K322A silencing mutations in the Fc region. It is.
• huIgG1si (Aragen, lot number BP-2211-018-6) is a human IgG1/kappa backbone anti-RSV mAb with an E269R/K322A silencing mutation in the Fc region.
- INX201J (Abzena, lot numbers JZ-0556-025-1, JZ-0556-027, JZ-0556-013) is a 8.0 drug/ INX201 antibody with antibody ratio (DAR). The linker/payload (J) is based on a previously reported linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker and a budesonide analogue payload (INX J-2).
• huIgG1si J (Abzena, lot number JZ-0556-025-2) is a huIgG1si antibody with a DAR of 8.0 conjugated through full modification of interchain disulfides with INX J linker/payload.
• INX201N (Abzena, lot number JZ-0556-028) is INX201 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX N) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-1).
• INX201O (Abzena, lot number JZ-0556-016-2) is INX201 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX O) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-4).
• INX201P (Abzena, lot number JZ-0556-016-1) is INX201 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-3).
- INX233 (ATUM, lot number 82276.1.a) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone that has L234A/L235A/E269R/K322A silencing mutations in the Fc region.
• INX233P (Abzena, lot number PP-0924-001-3) is INX233 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
- INX231 (ATUM, lot number 72928.1.a) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone that has L234A/L235A/E269R/K322A silencing mutations in the Fc region.
• INX231P (Abzena, lot number JZ-0556-017-1) is INX201 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
• INX231R (Abzena, lot number PP-0924-001-2) is INX231 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX R) consists of a neutral protease-sensitive linker and a budesonide analog payload (INX-SM-3).
• INX231S (Abzena, lot number PP-0920-014-1) is INX231 with a DAR of 6.9, conjugated through complete modification of the interchain disulfide. The linker/payload (INX S) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-24).
• INX231V (Abzena, lot number PP-0920-014-2) is INX231 with a DAR of 7.8, conjugated through complete modification of the interchain disulfide. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX231W (Abzena, lot number PP-0920-014-3) is INX231 with a DAR of 7.5 conjugated through complete modification of the interchain disulfide. The linker/payload (INX W) consists of a positively charged protease-sensitive linker and a budesonide analog payload (INX-SM-3).
• INX231J (Abzena, lot number JZ-0556-013-1) is an INX231 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (J) consists of a negatively charged protease sensitive linker and a budesonide analog payload (INX J-2).
• INX234J (Abzena, lot number JZ-0556-013-3) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (J) consists of a negatively charged protease sensitive linker and a budesonide analog payload (INX J-2).
• INX240J (Abzena, lot number JZ-0556-013-3) is an INX240 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (J) consists of a negatively charged protease sensitive linker and a budesonide analog payload (INX J-2).
• INX234P (Abzena, lot number HA-0853-02) is INX234 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
• INX234A3 (Abzena, lot number PP-0924-023-1) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (INX A3) consists of a positively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX234A4 (Abzena, lot number PP-0924-023-2) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 7.9. The linker/payload (INX A4) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-43).
• INX234T (Abzena, lot number PP-0924-023-3) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (INX T) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-3) and is phosphorylated.
• INX234V (Abzena, RJS-1054-003) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX234A5 (Abzena, lot number RJS-1054-002) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 7.9. The linker/payload (INX A5) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-44).
• INX234A11 (Abzena, lot number RJS-1054-001) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 8.0. The linker/payload (INX A11) consists of a negatively charged protease-sensitive linker (Asn/gly) and a budesonide analog payload (INX-SM-32).
• INX231A7 (Abzena, lot number RJS-1054-007-001) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 7.8. The linker/payload (INX A7) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32) and is phosphorylated.
• INX231A12 (Abzena, lot number RJS-1054-007-002) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 6.99. The linker/payload (INX A12) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-25) and is phosphorylated.
• INX231A23 (Abzena, lot number RJS-1054-006-001) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 7.34. The linker/payload (INX A23) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-25).
• INX234A13 (Abzena, lot number RJS-1054-007-003) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 5.82. The linker/payload (INX A13) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-34).
• INX234A1 (Abzena, lot number RJS-1054-004) is an INX234 antibody conjugated through complete modification of interchain disulfides and has a drug/antibody ratio (DAR) of 7.6. The linker/payload (INX A1) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-35).
• Target BP (Abzena PP-0924-031) is an anti-mouse Target B antibody with a DAR of 8.0, conjugated through complete modification of interchain disulfides. This antibody binds immune cell-specific antigens other than VISTA, is expressed on different immune cell types, and is an internalizing antibody. The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
• INX201V (Abzena, lot number SCG-1120-012-1) is INX201 with a DAR of 7.86, conjugated through complete modification of the interchain disulfide. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX234A9 (Abzena, lot number AF-1114-007-1) is an INX234 antibody conjugated via interchain disulfide modification with a drug/antibody ratio (DAR) of 7.3. The linker/payload (INX A9) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-46).

Antibodies were diluted in PBS and injected intraperitoneally (i.p.) in a volume of 0.2 ml to deliver the indicated doses.

Dexamethasone Dexamethasone Sterile Injection from Phoenix, NDC57319-519-05, was diluted in PBS and administered as described via intraperitoneal injection.

LPS
LPS was obtained from AMSBIO (number 9028). It was administered to mice at 0.5 mg/Kg.

Mice hVISTA mice were bred at the Center for Comparative Medicine and Research at Dartmouth. All experiments were performed on female mice enrolled between 9 and 15 weeks of age.

Blood Sampling and Preparation Peripheral blood was collected from the retroorbital space using a glass Pasteur pipette that was first rinsed with heparin to prevent clotting. Blood was then centrifuged at 550 rcf for 5 minutes and plasma was collected and stored at -80°C until cytokine analysis.

Plasma Cytokine Analysis Cytokine Analysis Using the Millipore Platform Cytokine analysis was performed on 25 μl of plasma using the Millipore mouse 5 or 7-plex platform. For ADC-INVIVO-30 and 35, the analysis was performed by the Immune Monitoring Laboratory (IML, a shared resource of the Dartmouth-Hitchcock Norris Cotton Cancer Center).

Cytokines included in the analysis were MIP-1α, TNFα, IL-1β, IL-12p40, and MIG, detected via ELISA as follows:
●BioLegend (Cat. No. 430904) ELISA MAX Deluxe Set Mouse TNF-α
●BioLegend (Cat. No. 431604) ELISA MAX Deluxe Set Mouse IL-12/IL-23 (p40)

ELISA was performed according to the manufacturer's attached protocol.

Cytokine data are censored if they are below the 20 pg/ml threshold for IL-12p40 and/or the 10 pg/ml threshold for TNF-α, indicating that the LPS injection has failed.

Cell Isolation After being euthanized, mice were injected intraperitoneally with 7 ml of PBS/0.5% BSA/2mM EDTA. After briefly massaging the peritoneum, a small incision was made and peritoneal lavage fluid was collected. PRMs were isolated using negative selection (Miltenyi kit, reference 130-110-434). Spleens were dissected and mechanically dissociated, and monocytes were isolated using negative selection (Stem Cell, EasySep™ Mouse CD11b Positive Selection Kit II).

RNA preparation and real-time PCR
Cell pellets from different tissues were resuspended in 0.4 ml RNeasy lysis buffer from the RNeasy Plus mini kit (Qiagen, PN: 74136) and homogenized 5 times with a 20G needle. RNA was isolated according to the manufacturer's instructions and eluted in 30 or 40 μl H ₂ O (RNase/DNase free). RNA concentration was assessed by UV spectroscopy using a Nanodrop.

Reverse transcription was performed using Taqman reverse transcription reagents (number N8080234) according to the manufacturer's instructions.

Quantitative real-time PCR was performed using the Taqman Master Mix 2X kit (number 4369016) and Taqman primers for mouse FKBP5 (Mm00487401_m1) and mouse HPRT (Mm446968_m1) as the housekeeping gene, using QuantStud from Applied Biosystems. Run on io3 did.

Ct data were converted to ΔCt (FKBP5 normalized to HPRT within the sample) and then to ΔΔCt (relative FKBP5 levels of treated samples vs. PBS control) to obtain Log2 fold change relative to PBS.

Results Experiment 1: Evaluation of In Vivo Efficacy of INX201J Efficacy in LPS-Induced Cytokine Release As shown in Figure 35, treatment with 10 mg/Kg INX201J significantly inhibits 2 mg/Kg Dex in controlling LPS-induced IL-12p40 release. showed similar effectiveness. It is important to note that 10 mg/kg of INX201J delivers a molar equivalent of 0.2 mg/Kg of GC payload, a dose where Dex has only partial efficacy.

This experiment further evaluated whether ADC requires longer processing times than free Dex. We showed improved efficacy when ADC was administered 17 hours before LPS injection when compared to 2 hours before LPS. No differences in cytokine responses were observed between 2 and 4 hours post-LPS, and in all subsequent studies plasma was collected only at the 2 hour time point. Figure 35 shows changes in IL-12p40 in peripheral blood 2 hours (left) and 4 hours (right) after LPS. Plasma concentrations measured using mouse multiplex. Administration: Dex (squares) was administered 2 hours before LPS stimulation at 0.02, 0.2, 2 and 5 mg/Kg, INX201J (circles) was administered at 10 mg/Kg to provide 0.2 mg/Kg of GC. Kg 2 hours or 17 hours before LPS injection. The PBS only group (gray filled triangles) represents baseline cytokine levels in the absence of stimulation. PBS+LPS (filled triangles) (SEM; n=5/group, unless technical failures are excluded from analysis; conventional one-way ANOVA compared to PBS+LPS group). Data show that administration of INX201J at 10 mg/Kg (delivering approximately 0.2 mg/Kg of GC payload) is similar to administration of dexamethasone at 2 or 5 mg/Kg in controlling LPS-induced IL-12p40 responses. Indicates that effectiveness has been demonstrated.

Experiment 2: INX201J dose response on LPS-induced cytokine release This experiment evaluated INX201J anti-inflammatory properties at higher dilutions. As shown in Figure 36, INX201J at 10 mg/Kg (0.2 mg/Kg payload) had efficacy comparable to that of Dex at 2 mg/Kg for all cytokines analyzed except MIG. while Dex at 0.2 mg/Kg had reduced efficacy compared to INX201J. INX201J showed some efficacy even when diluted to 0.06 and 0.02 mg/Kg payload.

We also tested the effectiveness of Dex when injected 17 hours before LPS, as was done with INX201J. More specifically, the experimental data in Figure 36 shows changes in cytokines in peripheral blood 2 hours after LPS. In these experiments, plasma concentrations were measured using a mouse 5-plex, and Dex was administered at 0.002, 0.02, 0.2, 2 mg/Kg for 2 hours before LPS stimulation (squares), or at 2 mg/Kg. INX201J (circle) was administered 17 hours before LPS injection at GC payloads of 0.02, 0.06, and 0.2 mg/Kg. . The PBS only group (gray filled triangles) represents baseline cytokine levels in the absence of stimulation. PBS+LPS (filled triangles) (SEM; n=5/group, unless technical failures are excluded from analysis; conventional one-way ANOVA compared to PBS+LPS group). As expected, the short half-life of Dex resulted in decreased efficacy and increased pharmacodynamic effects of INX201J on cytokine production in that group compared to the group administered 2 hours before LPS. suggests that it is possible. Data show that INX201J at 10 mg/Kg (delivering approximately 0.2 mg/Kg GC payload) and 2 mg/Kg in the control of LPS-induced MIP-1α, TNFα, IL-1β, IL-12p40, and MIG responses. It shows that it had effectiveness comparable to Dex. In comparison, Dex at 0.2 mg/Kg had only limited efficacy.

Experiment 3: INX201J dose response on LPS-induced cytokine release This experiment compared the efficacy of INX201J at GC payloads of 0.2 and 0.06 mg/Kg versus 2 and 0.2 mg/Kg of free Dex. Specifically, the experiment in Figure 37 shows changes in TNFα in peripheral blood 2 hours after LPS. TNFα plasma concentrations were measured using ELISA. Administration: Dex was administered 2 hours before LPS stimulation at 0.2 and 2 mg/Kg (squares), INX201J (circles) at 0.06 and 0.2 mg/Kg GC payload 17 hours after LPS injection. administered before. The PBS group (filled triangles) received PBS 2 hours before LPS. The IgG1siJ (G1siJ) group (triangles) received human IgG1 silently conjugated to GC in a payload of 0.2 mg/Kg 17 hours before LPS. (SEM; n=5/group, except when technical failures are excluded from analysis; normal one-way ANOVA compared to PBS group).

As shown from the data in Figure 37, INX201J at 0.2 mg/Kg GC payload was equally effective in controlling the TNFα response to LPS versus Dex at 2 mg/Kg. INX201J at 0.06 mg/Kg GC payload still showed higher efficacy than Dex at 0.2 mg/Kg. A control group injected with control human IgG1 silent conjugated with the same payload showed some efficacy in preventing TNFα upregulation. Dose-response studies showed improved/increased efficacy when GC payload was delivered via ADC INX201J, with 0.2 mg/Kg free Dex effective in preventing LPS-induced upregulation of TNFα However, molar equivalents of GC payload delivered via the ADC showed high potency. Note: Human IgG1 silent control conjugated to GC (same linker and GC payload as INX201J) appeared to have a weak effect on LPS-induced upregulation of TNFα.

Experiment 4: Evaluation of the in vivo efficacy of INX201J versus dexamethasone in LPS-induced cytokine release The experiment in Figure 38 shows the changes in TNFα 2 hours after LPS in peripheral blood using different ADCs, and TNFα plasma concentrations were determined by ELISA. Dex was administered 2 hours before LPS stimulation at 0.2 and 2 mg/Kg (squares), and INX201J (circles) and INX201N (inverted triangles) were administered at GC payload of 0.2 mg/Kg, respectively. Administered 17 hours before LPS injection, the PBS group received PBS 2 hours before LPS (filled triangles). (SEM; n=5/group, except when technical failures are excluded from analysis; normal one-way ANOVA compared to PBS group).

As shown from the data in Figure 38, and further observed in the experiments described above, INX201J at 0.2 mg/Kg GC payload is superior to Dex at 2 mg/Kg in controlling LPS-induced cytokine responses. had similar efficacy. In addition, the data show that INX201N has no efficacy in controlling TNFα responses, possibly due to inefficient cleavage or microaggregate formation of the released products of this linker/payload in vivo. to show that INX201J, when administered at 10 mg/Kg (~0.2 mg/Kg GC payload), prevented LPS from inducing TNFα upregulation with a potency comparable to Dex at 2 mg/Kg. Conversely, INX201N showed no efficacy in that model.

Experiment 5: Evaluation of in vivo efficacy of INX231J, INX234J, and INX240J versus INX201J in LPS-induced cytokine release Three different anti-VISTA antibodies conjugated to the same INX J payload were further evaluated. Specifically, the experiment in Figure 39 shows changes in TNFα (left) and IL-12p40 (right) in peripheral blood 2 hours after LPS, with cytokine plasma concentrations measured by ELISA and PBS (filled (circles), INX201J (squares), INX231J (triangles), INX234J (diamonds), and INX201P (inverted triangles) were administered 17 hours before LPS injection in a GC payload of 0.2 mg/Kg (SEM; n= 5/group; conventional one-way ANOVA compared to PBS group).

As shown from the data in Figure 39, INX231J, INX234J, and INX240J showed similar efficacy as INX201J in regulating LPS-induced cytokine responses. All ADCs administered at 10 mg/Kg (delivering approximately 0.2 mg/Kg GC payload) showed comparable efficacy to INX201J in controlling LPS-induced TNFα and IL-12p40 responses.

Experiment 6: Evaluation of the in vivo efficacy of INX201O and INX201P vs. INX201J in LPS-induced cytokine release In the experiment of Figure 40, different ADCs were administered at 10 mg/Kg, delivering a payload of 0.2 mg/Kg and targeting specific cytokines. We evaluated their effectiveness. Specifically, Figure 40 shows changes in TNFα (left) and IL-12p40 (right) in peripheral blood 2 hours after LPS, cytokine plasma concentrations were measured by ELISA, and PBS (filled triangles); INX201J (circles), INX201O (squares), and INX201P (diamonds) were administered 17 hours before LPS injection in a GC payload of 0.2 mg/Kg (SEM; unless technical failures excluded from analysis. , n=5/group; conventional one-way ANOVA compared to PBS group).

As shown by the data in Figure 40, INX201P showed similar efficacy as INX201J in controlling LPS-induced cytokine responses, whereas INX201O was less effective. INX201P was as effective as INX201J at 10 mg/Kg (delivering approximately 0.2 mg/Kg of GC payload) in controlling LPS-induced TNFα and IL-12p40 responses. In comparison, INX201O showed reduced activity.

Experiment 7: Evaluation of in vivo efficacy of INX201O and INX201P versus INX201J in LPS-induced cytokine release - dose response study In the experiment of Figure 41, cytokine plasma concentrations were determined by ELISA and measured in PBS, INX201J (circles), INX201O (squares), and INX201P (diamonds) were administered 17 hours before LPS injection in 0.2 mg/Kg GC payload, respectively (SEM; n = 5/group, unless technical failure excluded from analysis; PBS group (regular one-way ANOVA compared to (black filled triangles)). Specifically, FIG. 41 shows changes in TNFα (right) and IL-12p40 (left) in peripheral blood 2 hours after LPS.

Similar to the results of the previous experiment 6, INX201O showed decreased efficacy compared to INX201J in these experiments. In contrast, both INX201P and INX201J showed similar efficacy in controlling IL-12p40 and TNFα responses to LPS. Also note that with a payload of 0.06 mg/Kg, there is still strong control of the cytokine response (Figure 41). In this repeat of previous experiments including dose-response comparisons, INX201P showed comparable efficacy as INX201J in controlling LPS-induced TNFα and IL-12p40 responses. INX 201O again showed reduced activity.

Experiment 8: Evaluation of in vivo efficacy of INX231R, INX233P vs. INX231P in LPS-induced cytokine release. shows the change in In these experiments, cytokine plasma concentrations were measured by ELISA, and all ADCs and PBS were administered 20 hours before LPS injection at GC payload of 0.2 mg/Kg (INX231P (squares), INX231R (triangles), INX233P (diamonds)) (SEM; n = 5/group, except when technical failures are excluded from the analysis; conventional one-way ANOVA compared to the PBS group (filled circles)).

As can be seen in Figure 42, INX231R (neutral dipeptide linker) showed little effect on IL-12p40 induction, but a significant reduction in TNFα after LPS injection. In contrast, INX233P had similar efficacy to INX231P (both have negatively charged dipeptide linkers). All ADCs were dosed at 10 mg/Kg, delivering a payload of 0.2 mg/Kg. INX231R and INX233P showed comparable efficacy as INX231P at 10 mg/Kg (delivering approximately 0.2 mg/Kg GC payload) in controlling LPS-induced TNFα and IL-12p40 responses.

Experiment 9: Evaluation of the in vivo efficacy of INX231R, INX201O, INX231S, INX231V, and INX231W versus INX231P in LPS-induced cytokine release The experiment in Figure 43 shows that TNFα (right) and IL-12p40 ( (left) shows changes in In these experiments, cytokine plasma concentrations were measured by ELISA. All ADCs and PBS were administered 20 hours before LPS injection at GC payload of 0.2 mg/Kg (INX231P (filled squares), INX231R (filled triangles), INX201O (filled diamonds), INX231S (circles). ), INX231V (squares), INX231W (triangles)) (SEM; n = 4/group, except for INX231S where two technical failures are excluded from the analysis; normal unidirectional compared to PBS group (filled circles) ANOVA showed non-significant data). Also, in this experiment, some ADCs had DAR below 8, so ADC dosing was adjusted to deliver a payload of 0.2 mg/Kg.

As observed in Experiment 8, INX231R was less effective than INX231P in these experiments, and INX231W behaved similarly, primarily affecting TNFα. Additionally, INX231S and INX231V showed similar efficacy as INX231P. Finally, as observed in the previous two experiments, INX201O showed reduced efficacy compared to other ADCs (Figure 43). INX231R, INX231S, INX231V, and INX231W showed comparable efficacy as INX231P at 10 mg/Kg (delivering approximately 0.2 mg/Kg GC payload) in controlling LPS-induced TNFα and IL-12p40 responses. In comparison, INX201O showed reduced activity. In addition, all ADCs tested except INX201O potently delivered their GC payloads, as demonstrated by FKBP5 transcriptional induction, over a pharmacodynamic range of at least 4 days.

Experiment 10: Comparison of the effects of INX231R, INX201O, INX231S, INX231V, and INX231W versus INX231P on FKBP5 transcription The experiment in Figure 44 shows FKBP5 transcriptional activation after ADC treatment in peritoneal residents 4 days after ADC treatment. In these experiments, ADCs were injected intraperitoneally on day 0, each delivering a GC payload of 0.2 mg/Kg, and PRMs were isolated on day 3. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus PBS control group (SEM, conventional one-way ANOVA compared to PBS group, n=4).

As shown herein, peritoneal resident macrophages (PRMs) are highly sensitive to ADCs, and GC effects on GC target FKBP5 can be measured by real-time quantitative PCR (RT-qPCR). Therefore, PRMs were isolated on day 3 after LPS treatment (day 4 after ADC administration), RNA was extracted, and RT-qPCR was performed for FKBP5. As shown from the data in Figure 44, all ADCs except INX201O induced strong FKBP5 transcription, with adequate delivery of GC payload and a pharmacodynamic range of at least 4 days). INX234P, INX234A3, and INX234A4 showed efficacy comparable to 10 mg/Kg (delivering approximately 0.2 mg/Kg of GC payload) in controlling LPS-induced TNFα and IL-12p40 responses. INX234T had a limited effect on TNFα. INX231R, INX231S, INX231V, and INX231W showed comparable efficacy as INX231P at 10 mg/Kg (delivering approximately 0.2 mg/Kg GC payload) in controlling LPS-induced TNFα and IL-12p40 responses. In comparison, INX201O showed reduced activity. In addition, all ADCs tested except INX201O potently delivered their GC payloads, as demonstrated by FKBP5 transcriptional induction, over a pharmacodynamic range of at least 4 days.

Experiment 11: Evaluation of in vivo efficacy of INX234P, INX234A3, INX234A4, INX234T, and Target BP on LPS-induced cytokine responses. and showed efficacy comparable to 10 mg/Kg (delivering approximately 0.2 mg/Kg of GC payload) in controlling IL-12p40 responses. INX234T had a limited effect on TNFα. In this experiment, we further evaluated ADC Target BP, an antibody directed against a murine version of a new target (Target B) conjugated with the INX P payload, and found that Target BP showed similar efficacy as INX234T. IL-12P40 was significantly reduced, but TNFα remained unchanged.

More specifically, FIG. 45 shows changes in TNFα (right) and IL-12p40 (left) in peripheral blood 2 hours after LPS. Cytokine plasma concentrations were measured by ELISA. All ADCs and PBS were administered 20 hours before LPS injection at GC payload of 0.2 mg/Kg (SEM; except INX234P, INX234A4, and INX234T where one technical failure was recorded per group, n= 5/group; conventional one-way ANOVA compared to PBS group). ADCs containing antibodies against another immune cell-specific antigen (other than VISTA) conjugated to the INX P payload showed similar efficacy to INX234T, with significantly reduced IL-12P40 but no change in TNFα .

Experiment 12: Evaluation of the efficacy of INX234V, INX234A5, INX234A11 versus PBS on short-term LPS-induced cytokine release. Changes are shown and cytokine plasma concentrations were measured by ELISA, all ADCs and PBS were administered 20 hours before LPS injection at 0.2 mg/Kg GC payload (INX234V (filled squares), INX234A5 (filled triangles), INX234A11 (filled diamonds)) (SEM; n=5/group; conventional one-way ANOVA compared to PBS (filled circles) group). The data in Figure 46 show that INX234V, INX234A5, and INX234A11 are as effective as 10 mg/Kg (delivering approximately 0.2 mg/Kg of GC payload) in controlling LPS-induced TNFα and IL-12p40 responses. It was shown that

Experiment 13: Evaluation of the efficacy of INX231A7, INX231A12, INX231A23; INX234A1, INX234A13 versus INX234V and PS on short-term LPS-induced cytokine release. (right) and IL-12p40 (left). In these experiments, cytokine plasma concentrations were measured by ELISA, and all ADCs and PBS were administered with a GC payload of 0.2 mg/Kg (INX234V (filled square), except for INX231A7, which was administered with a payload of 0.08 mg/Kg. ) administered 20 hours before LPS injection in ) (SEM; n = 5/group; Conventional one-way ANOVA compared to PBS group).

As shown by the results in Figure 47, INX234V, INX231A12, and INX231A23 showed similar efficacy in controlling the production of IL-12p40 and TNFα. INX2341A7 still drove significant changes in IL-12p40 and was close to significant (P=0.052) but less potent for TNFα. (Note, however, that INX231A7 was administered at a payload of 0.08 mg/Kg). Also, both INX234A1 and INX234A13 had no effect on LPS-induced cytokine production. INX234V, INX231A12, INX231A23, and INX234A1 showed efficacy comparable to 10 mg/Kg (delivering approximately 0.2 mg/Kg GC payload) in controlling LPS-induced TNFα and IL-12p40 responses. INX231A7 also showed comparable efficacy while administered at a payload of 0.08 mg/Kg. Both INX234A13 and INX234A11 showed no efficacy

Experiment 14: Evaluation of the efficacy of INX234P, INX234A9, INX201V versus PBS on short-term LPS-induced cytokine release. (Left) shows the change. In these experiments, cytokine plasma concentrations were measured by ELISA, and all ADCs and PBS were administered 20 hours before LPS injection at 0.2 mg/Kg GC payload (SEM; Excluding one sample that needed to be censored and two samples in the INX234A9 group for technical reasons, n=5/group; normal one-way ANOVA compared to the PBS group).

As shown from the data in Figure 48, INX234A9 and INX201V appear to be less potent than INX234P in preventing cytokine responses; however, the reduced efficacy is driven by one outlier point (same animal showed no effect of treatment on both cytokines, which may be due to injection failure).

Conclusion As disclosed herein, different ADCs containing different anti-VISTA antibodies that bind to VISTA at physiological pH and have shorter PKs, different complementarity determining regions (CDRs), and different GC payloads have been synthesized. has been done.

Data show that immune cells used each of INX201, INX231, INX234, INX240, or INX233 to target GC delivery:
• Efficiently reduces LPS-induced cytokine responses • Delivering approximately 10 times lower doses of GC on a mg/Kg payload basis allows similar efficacy.
• May result in increased duration of pharmacodynamics of GC.

In addition, conjugates similar to the INX P linker payload were evaluated in which the charge of the dipeptide linker was changed. These include positive charges (INX W), neutral charges (INX R), and negative charges (INX P). These results showed:
• In this model, both positively charged INX W and neutral INX R were effective, but negatively charged INX P was more potent.
• All dipeptide linker variants (positive, negative, neutral) exhibited a pharmacodynamic range of at least 4 days, as demonstrated by high level expression of the GC reporter FKBP5.

In addition to the initial INX J linker/payload that varied the structure of the payload, conjugates with four budesonide analog linkers/payloads, INX N, INX O, INX P, and INX V, were further evaluated. These results showed:
• The conjugated INX N linker/payload had no efficacy in the short-term LPS activation model, likely reflecting lack of efficient cleavage or microaggregate formation of the released product of INX N.
• Conjugated INX O linker/payload had an effect in this model, but showed reduced potency compared to conjugated INX J, INX P, or INX V linker/payload.
- Conjugate INXP showed similar efficacy as conjugated INX J and INX V linkers/payloads.
• Conjugate INX P, INX V, and INX J linker payloads exhibited a pharmacodynamic range of at least 4 days, as demonstrated by high level expression of the GC reporter gene FKBP5.
● Certain payload changes can be tolerated without interrupting efficacy.

A conjugate with a fluocinolone acetonide analog (INX S) was evaluated in comparison to its budesonide analog counterpart (INX P). These results showed:
●INX231P and INX231S had similar efficacy.
• Both INX231P and INX231S exhibited a pharmacodynamic range of at least 4 days, as demonstrated by high level expression of the GC reporter gene FKBP5.
• INX234P, INX234A3, and INX234A4 had similar efficacy, but INX234T showed more limited (and non-significant) control of LPS-induced TNFa production.
• INX234V, INX234A5, and INX234A11 showed similar efficacy in controlling LPS-induced IL-12p40 and TNFa production.
• INX231A7, INX231A12, and INX231A23 showed similar potency as INX234V in controlling LPS-induced IL-12p40 and TNFa production, whereas INX234A1 and INX234A13 had no effect.
• INX234V, INX231A12, INX231A23, and INX234A1 showed efficacy comparable to 10 mg/Kg (delivering approximately 0.2 mg/Kg GC payload) in controlling LPS-induced TNFα and IL-12p40 responses. INX231A7 also showed comparable efficacy while administered at a payload of 0.08 mg/Kg. In contrast, both INX234A13 and INX234A11 showed no efficacy.

More importantly, when an exemplary payload according to the invention, the INX P payload, is conjugated with an antibody that binds to a distinct target (not VISTA), including the murine ortholog of the antigen termed target B, It was shown to preserve its potency when delivered to cells. This result is similar to what was seen when the INX P payload was conjugated to an antibody that binds another immune cell antigen, and when this same payload was conjugated to an anti-VISTA antibody. Indicates that it was captured efficiently. On this basis, the payload according to the present invention may contain other (non-VISTA) immune cell antigens, such as B, T, NK, bone marrow, macrophages, Tregs, neutrophils, among other immune cell types. It is anticipated that the payloads according to the invention could be conjugated to other antibodies that bind to other antigens expressed by one or more types of immune cells, including dendritic cells and dendritic cells, and that the payloads according to the invention could be used to effect these immune cells. will be delivered accordingly.

We also demonstrated that two conjugates with a linker payload containing a spirocenter near the break point, INX234A9 and INX201V, appeared to be less effective than INX234P in regulating LPS-induced IL-12p40 and TNFa production. , the reduced efficacy is driven by possible outlier data points (the same animals showed no treatment effect for both cytokines, so these efficacy differences could be due to injection failure) .

References 1-Vermeer et al. (2003) Glucocorticoid-induced increase in lymphocytic FKBP51 messenger ribonucleic acid expression: a potential marker for glue cocorticoid sensitivity, potency, and bioavailability. J Clin Endocrinol Metab. Jan;88(1):277-84
2-McPherson MJ, et al. (2017) Glucocorticoid receptor agonist and immunoconjugates thereof, US15/611,037

Example 8: Anti-VISTA antibody drug conjugates have limited effects on non-VISTA expressing cells The effects of exemplary ADCs on non-target (non-VISTA) expressing cells are described in this example and shown in FIG. It was evaluated in the experiments shown in Experiments. The purpose of these studies was to verify the targeting specificity of the ADCs of the invention to VISTA expressing cells/tissues compared to free dexamethasone (Dex). To monitor/confirm GC delivery and activity, transcriptional activation of FKBP5, a sensitive and early GC response gene, was measured by quantitative real-time PCR (qRT-PCR) (Vermeer et al., (2003) Glucocorticoid- induced increase in lymphocytic FKBP51 messenger ribonucleic acid expression: a potential marker for glucocorticoid sensitivity J Clin Endocrinol Metab. Jan; 88(1):277-84). In these experiments, which are disclosed in detail below, INX201J or free Dex was delivered in vivo via intraperitoneal (i.p.) injection, followed by injection of VISTA-expressing splenocytes, as well as non-VISTA from liver, brain, and adrenal glands. Expressing cells were isolated. RNA was then extracted and FKBP5 transcript levels were evaluated.

Materials and Methods Two hours before mice were euthanized and cells isolated, Dex was injected intraperitoneally, which corresponds to the peak of FKBP5 induction. INX201J was injected 20 hours before mice were euthanized and cells isolated to provide sufficient time for peak ADC treatment and FKBP5 induction. A control group injected with PBS only was included to define the FKBP5 transcriptional baseline.
Test drug and dosage Antibody INX201 (Aragen, lot number BP-3200-019-6) is a humanized anti-human VISTA antibody with a human IgG1/kappa backbone that has L234A/L235A/E269R/K322A silencing mutations in the Fc region. It is.
- INX201J (Abzena, lot numbers JZ-0556-025-1, JZ-0556-027, JZ-0556-013) is a 8.0 drug/ INX201 antibody with antibody ratio (DAR). The linker/payload (J) is based on a previously reported linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker and a budesonide analogue payload (INX J-2).

These antibodies were diluted in PBS and injected intraperitoneally (i.p.) in a volume of 0.2 ml to deliver the indicated doses.

Dexamethasone Dexamethasone Sterile Injection from Phoenix, NDC57319-519-05, was diluted in PBS and administered as described via intraperitoneal injection.

Mice hVISTA KI mice were bred at the Center for Comparative Medicine and Research at Dartmouth. All experiments were performed on female mice enrolled between 9 and 15 weeks of age.

Cell Isolation After euthanasia, the spleen, liver, adrenal glands and brain were dissected and mechanically dissociated. After passing through a 40 μm filter, the cell pellet was resuspended in RNA lysis buffer (see below).

RNA preparation and real-time PCR
Cell pellets from different tissues were resuspended in 0.4 ml RNeasy lysis buffer from the RNeasy Plus mini kit (Qiagen, PN: 74136) and homogenized 5 times with a 20G needle. RNA was isolated according to the manufacturer's instructions and eluted in 30 or 40 μl H ₂ O (RNase/DNase free). RNA concentration was assessed on a Nanodrop.

Reverse transcription was performed using Taqman reverse transcription reagents (number N8080234) according to the manufacturer's instructions. Quantitative real-time PCR was performed using the Taqman Master Mix 2X kit (number 4369016) and Taqman primers for mouse FKBP5 (Mm00487401_m1) and mouse HPRT (Mm446968_m1) as the housekeeping gene, using QuantStud from Applied Biosystems. Run on io3 did.

Ct data were converted to ΔCt and ΔΔCt or Log2 fold change was converted to PBS.

Results Briefly, liver endothelial cells were isolated from hVISTA KI mice using the Liver Dissociation Kit and Liver Endothelial Cell Isolation Kit from Miltenyi (130-105-807 and 130-092-007, respectively). As shown in the experiment in Figure 49, CD45-negative (non-immune) CD31-positive (endothelial) cells exhibit high levels of VISTA expression (red line VISTA; solid gray, no antibody). Specifically, Figure 49 shows that VISTA was isolated from liver endothelial cells, specifically hVISTA knock-in mouse liver, and either stained with anti-human VISTA (red line, shifted to the right) or unstained (filled). CD45-CD31+ non-immune endothelial cells (gray).

In the experiment shown in Figure 50, the effects of INX201J versus Dex on non-VISTA expressing tissues (adrenals, brain, and liver) and VISTA-expressing spleen in female hVISTA KI mice was evaluated. Specifically, FKBP5 transcriptional activation after INX201J injection in adrenal gland, brain, liver, and spleen is shown in FIG. INX201J effects were measured 20 hours after a single intraperitoneal injection at 0.3, 3, and 10 mg/Kg (delivering payloads of 0.006, 0.06, and 0.2 mg/Kg, respectively). Dex effects were measured 2 hours after a single intraperitoneal injection at 0.2 or 2 mg/Kg. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus the mean of the PBS control group. (n=4 mice/group, normal one-way ANOVA compared to PBS only group).

As can be seen from the results in Figure 50, no signal above baseline was detected in the adrenal glands and brain after INX201J injection, but 2 mg/Kg of Dex caused a slight increase in FKBP5 transcripts in the adrenal glands and a slight increase in FKBP5 transcripts in the brain. brought about a strong increase. In the liver, FKBP5 was detected at levels similar to INX201J or Dex at 0.2 mg/Kg payload and elevated at 2 mg/Kg with Dex.

Furthermore, a clear dose-dependent induction by INX201J was observed in the spleen, with a 10-fold increase in FKBP5 signal with INX201J at 0.2 mg/Kg payload when compared to Dex at 0.2 mg/Kg payload. In contrast, comparable responses with Dex were achieved only at 2 mg/Kg.

Conclusion Data show that INX201J at 3 and 10 mg/Kg (0.06 and 0.2 mg/Kg payload) induces FKBP5 expression in VISTA-expressing splenocytes, but not in adrenal gland or brain (Figure 49) . In the liver, INX201J when administered at 3 and 10 mg/Kg (payload of 0.06 and 0.2 mg/Kg) was significantly reduced, likely due to the abundance of immune cells in this tissue and robust VISTA expression in liver endothelial cells. and slightly induce FKBP5 (Figure 50). In contrast, Dex at a treatment dose of 2 mg/Kg induced FKBP5 induction in splenocytes, and this same dose induced robust levels of FKBP5 in the brain and liver, and modest levels of FKBP5 in the adrenal glands. .

Example 9: In vitro efficacy of steroid payloads and antibody glucocorticoid conjugates of the invention in human peripheral blood mononuclear cells This example demonstrates the in vitro efficacy of different steroid payloads in human peripheral blood mononuclear cell steroids in an in vitro model of inflammation. Efficacy was evaluated. The presence of LPS results in PBMC proliferation and cytokine release (Jansky, L., Reymanova, P., & Kopecky, J. (2003), “Dynamics of cytokine production in human peripheral blood mon onuclear cells stimulated by LPS, or infected by Borrelia”, Physiological Research, 52(5), 593-5981).

Materials and Methods Methods The efficacy of novel steroids was evaluated in a model of LPS-stimulated human peripheral blood mononuclear cells (PBMCs). PBMCs stimulated in this assay produce multiple pro-inflammatory cytokines. Efficacy of steroids was judged in these studies by their ability to reduce stimulation-related cytokine expression in a dose-dependent manner compared to untreated at 24 hours.

The aim of this study was to characterize an in vitro model of inflammation in which a novel glucocorticoid produced with ImmuNext, identified as INX-SM-GC, was tested as a free small molecule or conjugated to an antibody via a peptide linker in wells. The purpose was to evaluate the effectiveness of Human PBMC produce several pro-inflammatory cytokines when stimulated with LPS, and this cytokine response can be dramatically inhibited by glucocorticoids (GC). The study used budesonide, a very potent and clinically relevant GC, as a comparator.

Materials and Methods Experimental Design In all experiments below, human PBMCs isolated from 1-2 healthy donors per study were stimulated with LPS to induce cytokine production.

Cells were treated with serially diluted GC or GC conjugate (1000-0.2 nM payload) to identify dose-dependent potency of individual drugs using budesonide as a positive control. LPS was added either immediately after addition of free payload or, in the case of conjugates, 4 hours after addition of conjugate.

Preliminary experiments identified IL-6 and IL-1b as highly GC-responsive cytokines. Therefore, after incubating PBMC with GC or antibody-conjugated GC for 24 hours, cell supernatants were collected and assessed for IL-6 and IL-1b cytokine levels via ELISA.
Reagent Test payload Budesonide: 10mM in DMSO
●INX-SM-1 (Abzena): 5mM in DMSO
●INX-SM-2 (Abzena): 2mM in DMSO
●INX-SM-3 (O2H): 10mM in DMSO
●INX-SM-53 (O2H): 10mM in DMSO (S stereoisomer of INX-SM-3)
●INX-SM-4 (O2H): 20mM in DMSO
●INX-SM-54 (O2H): 10mM in DMSO (S stereoisomer of INX-SM-4)
●INX-SM-6 (O2H): 10mM in DMSO
●INX-SM-56 (O2H): 10mM in DMSO (S stereoisomer of INX-SM-6)
●INX-SM-7 (O2H): 2mM in DMSO
●INX-SM-9 (O2H): 2mM in DMSO
●INX-SM-10 (O2H): 2mM in DMSO
●INX-SM-13 (O2H): 2mM in DMSO
●INX-SM-24 (O2H): 2mM in DMSO
●INX-SM-31 (O2H): 2mM in DMSO
●INX-SM-32 (O2H): 2mM in DMSO
●INX-SM-33 (O2H): 2mM in DMSO
●INX-SM-35 (O2H): 2mM in DMSO
●INX-SM-74 (O2H): 2mM in DMSO (S stereoisomer of INX-SM-24)
●INX-SM-43 (O2H): 20mM in DMSO
●INX-SM-44 (O2H): 20mM in DMSO
●INX-SM-45 (O2H): 20mM in DMSO
●INX-SM-46 (O2H): 20mM in DMSO
●INX-SM-36 (O2H): 20mM in DMSO
●INX-SM-37 (O2H): 20mM in DMSO
●INX-SM-J2 (O2H): 20mM in DMSO
●INX-SM-14 (O2H): 20mM in DMSO
●INX-SM-15 (O2H): 20mM in DMSO
●INX-SM-17 (O2H): 20mM in DMSO
●INX-SM-34 (O2H): 20mM in DMSO
●INX-SM-40 (O2H): 20mM in DMSO
●INX-SM-47 (O2H): 20mM in DMSO
●INX-SM-49 (O2H): 20mM in DMSO
●INX-SM-18 (O2H): 20mM in DMSO
●INX-SM-32 (O2H): 20mM in DMSO
●INX-SM-48 (O2H): 20mM in DMSO
●INX-J2 (INX-SM-J2) (O2H): 20mM in DMSO
- Dexamethasone (Sigma, catalog number D4902): 20mM in DMSO
Antibody Conjugate INX231J (Abzena, lot number JZ-0556-013-1) is conjugated through complete modification of interchain disulfides, with a drug/antibody ratio (DAR) of 8.0. It is INX231. The linker/payload (INX J) is based on the published linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker and a budesonide analogue payload (INX J2).
• INX231P (Abzena, lot number JZ-0556-017-1) is INX231 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
• INX231V (Abzena, lot number PP-0920-014-2) is INX231 with a DAR of 7.8, conjugated through complete modification of the interchain disulfide. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX234P (Abzena, lot number JZ-0556-017-2) is INX234 with a DAR of 8.0 conjugated through complete modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
• INX234A4 (Abzena, lot number PP-0924-023-2) is an INX234 antibody with a DAR of 7.9 conjugated via interchain disulfide modification. The linker/payload (INX A4) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-43).
• INX231S (Abzena, lot number PP-0920-014-1) is INX231 with a DAR of 6.9, conjugated through complete modification of the interchain disulfide. The linker/payload (INX S) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-24).
• INX234T (Abzena, lot number PP-0924-023-3) is an INX234 antibody with a DAR of 8.0 conjugated through complete modification of interchain disulfides. The linker/payload (INX T) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-3) and is phosphorylated.
• INX234A3 (Abzena, lot number PP-0924-023-1) is an INX234 antibody with a DAR of 8.0 conjugated through complete modification of interchain disulfides. The linker/payload (INX A3) consists of a positively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
●INX201J (Abzena, lot numbers: JZ-0556-025-1, JZ-0556-027, JZ-0556-013) is a linker/payload via complete modification of interchain disulfides with a DAR of 8.0. Conjugated INX201 antibody. The linker/payload (INX J) is based on the published linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker and a budesonide analogue payload (INX J2).
• INX201L (Abzena, lot number JZ-0556-026-1) is an INX201 antibody with a DAR of 8.0 conjugated through complete modification of interchain disulfides. The linker/payload (INXL) is based on the published linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker and a budesonide analogue payload (INX J-2), which is phosphorylated.
• INX234A11 (Abzena, lot number RJS-1054-001) is an INX234 antibody with a DAR of 8.0 conjugated through complete modification of interchain disulfides. The linker/payload (INX A11) consists of a negatively charged protease-sensitive linker (Asn/gly) and a budesonide analog payload (INX-SM-32).
• INX234V (Abzena, lot number RJS-1054-003) is an INX234 antibody with a DAR of 8.0 conjugated through complete modification of interchain disulfides. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX234A5 (Abzena, lot number RJS-1054-002) is an INX234 antibody with a DAR of 7.9 conjugated via interchain disulfide modification. The linker/payload (INX A5) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-44).
• INX231A23 (Abzena, lot number RJS-1054-006-001) is an INX231 antibody with a DAR of 7.34, conjugated via interchain disulfide modification. The linker/payload (INX A23) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-25).
• INX231A12 (Abzena, lot number RJS-1054-007-002) is an INX231 antibody with a DAR of 6.99, conjugated via interchain disulfide modification. The linker/payload (INX A12) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-25) and is phosphorylated.
• INX231A7 (Abzena, lot number RJS-1054-007-001) is an INX231 antibody with a DAR of 7.8, conjugated via interchain disulfide modification. The linker/payload (INX A7) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32) and is phosphorylated.
• INX234A13 (Abzena, lot number RJS-1054-007-003) is an INX234 antibody with a DAR of 5.82, conjugated via interchain disulfide modification. The linker/payload (INX A13) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-34).
• INX234A1 (Abzena, lot number RJS-1054-004) is an INX234 antibody with a DAR of 7.6, conjugated via interchain disulfide modification. The linker/payload (INX A1) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-35).
-INX1302 (ATUM, lot number 78597.2.a) is an Fc silent anti-human target B antibody.
INX1302P (Abzena, lot number PP-0924-019-002) is an Fc silent anti-human target B antibody, INX1302, with a DAR of 8.0, conjugated via complete modification of the interchain disulfide. . The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
• INX1400P (Abzena, lot number PP-0924-019-001) is an Fc silent anti-human Target A antibody with a DAR of 8.0, conjugated through complete modification of interchain disulfides. The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
• INX234J (Abzena, lot number JZ-0556-013-2) is an INX234 antibody with a DAR of 8.0 conjugated through complete modification of the interchain disulfides. The linker/payload (INX J) is based on the published linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker and a budesonide analogue payload (INX J2).
• INX234A9 (Abzena, lot number AF-1114-007-1) is an INX234 antibody with a DAR of 7.3 conjugated via interchain disulfide modification. The linker/payload (INX A9) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-46).
• INX201V (Abzena, lot number SCG-1120-012-1) is an INX201 antibody with a DAR of 7.86, conjugated via interchain disulfide modification. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX231V (Pearl River, lot number 101-1_02) is an INX231 antibody with a DAR of 3.66, conjugated via interchain disulfide modification. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
• INX201P (Pearl River, lot number 101-2_02) is an INX201 antibody with a DAR of 4.52, conjugated via interchain disulfide modification. The linker/payload (INX P) consists of a negatively charged protease sensitive linker (INX-SM-3) and a budesonide analog payload.
• INX201A23 (Pearl River, lot number 101-3_02) is an INX201 antibody with a DAR of 4.34 conjugated via interchain disulfide modification. The linker/payload (INX A23) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-25).
• INX201A23 (Pearl River, lot number 101-4_01) is an INX201 antibody with a DAR of 7.71, conjugated via interchain disulfide modification. The linker/payload (INX A23) consists of a negatively charged protease-sensitive linker and a fluocinolone acetonide analog payload (INX-SM-25).
• INX201V (Abzena, lot number SCG-1120-032-1) is an INX201 antibody with a DAR of 3.95, conjugated via interchain disulfide modification. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker and a budesonide analog payload (INX-SM-32).
Cell culture medium ●RPMI 1640 without L-glutamine (VWR, catalog number 16750-084)
●Penicillin/Streptomycin/Glutamine (ThermoFisher, catalog number 10378016)
●1M Hepes (Gibco, catalog number 15630-080)
●Human AB serum (Valley Biomedical, catalog number HP1022HI)
Other reagents Lipopolysaccharide from Escherichia coli O111:B4 (Sigma, Cat. No. L2630)
●Ficoll-Paque Plus (GE Healthcare, catalog number 17-1440-03)
ELISA kit Human IL-6 ELISA MAX Deluxe (Biolegend, catalog number 430504)
●Human IL-1β ELISA MAX Deluxe (Biolegend, catalog number 437004)

PBMC Preparation Human PBMC were isolated under sterile conditions from apheresis cones obtained from the Dartmouth Hitchcock Medical Center's Blood Donor Program from anonymized healthy human donors.

Blood was transferred to a 50 ml Falcon tube and diluted to 30 ml with PBS. 13 ml of Ficoll-Paque Plus (Sigma Aldrich) was slowly layered under the blood and the tube was centrifuged at 850 x g for 20 min at room temperature with gentle acceleration and no brake.

Mononuclear cells were collected from the plasma/Ficoll interface, resuspended in 50 ml of PBS, and centrifuged at 300×g for 5 minutes. Cells were resuspended in PBS and counted.

Assay Protocol Isolated PBMCs were resuspended in RPMI 1640 containing 10% human A/B serum, 10mM Hepes, 1x penicillin/streptomycin/L-glutamine (assay medium).

Cells were plated in flat-bottomed 96-well plates at a final concentration of 150,000 cells/well, and technical replicates were performed for each condition.

Test drugs were serially diluted in assay medium and added at final concentrations of 1,000 nM to 1 nM or 0.2 nM, depending on the assay or as an untreated control.

LPS stimulation was added at a final concentration of 1 ng/ml. For delayed LPS studies, LPS was added 4 hours after adding the antibody drug conjugates (INX231J, INX231P, INX231V).

The cells were placed in a 37°C, 5% CO2 incubator for 24 hours, and then the supernatant was collected.
Human IL-1β and IL-6 ELISA kits were used with supernatants according to the manufacturer's protocol.
All graphs were created with GraphPad (Prism).

Results Experiment 1: Evaluation of the inhibitory effects of steroid payloads INX-SM-3, INX-SM-53, INX-SM-4, INX-SM-54, and INX-SM-1 on cytokine production of LPS-stimulated human PBMCs. In the experiments presented in 51, the anti-inflammatory potency of the novel INX-GC payloads INX-SM-3, INX-SM-4, INX-SM-1, INX-SM-53, and INX-SM-54 was evaluated. PBMC from one donor were tested. As shown in Figure 51, INX-SM-3, INX-SM-4, and INX-SM-1 inhibited both IL-1β and IL-6 production, and INX-SM-3 inhibited both IL-1β and IL-6 production. It appears to be the most potent compound of the three. In contrast, the S stereoisomers at the acetal position, -INX-SM-53 and INX-SM-54, showed no inhibition.

From the data in Figure 51, it can be seen that INX-SM-3, INX-SM-4, and INX-SM-1 inhibit IL-1β (left) and IL-6 (right) production. Cytokine levels were measured over 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-1 nM), with <1 nM no treatment control plotted on the x-axis on a logarithmic scale. , n=1 donor and standard deviations were plotted from technical duplicates.

Experiment 2: Evaluate the inhibitory effects of steroid payloads INX-SM-6 and INX-SM-56 on cytokine production of LPS-stimulated human PBMCs, and evaluate the inhibitory effects of INX-SM-1, INX-SM-3, and INX-SM-4. The experiments in Figure 52 confirm the anti-inflammatory efficacy of the novel glucocorticoid payloads INX-SM-3, INX-SM-4, INX-SM-1, and additional compounds INX-SM-6 and INX -Efficacy of SM-56 was evaluated. PBMC from one donor were tested. Specifically, in Figure 52, cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-1 nM), and <1 nM are plotted on the x-axis on a logarithmic scale. No treatment controls were plotted, n=1 donor, and standard deviations were plotted from technical duplicates.

As shown in Figure 52, INX-SM-1, INX-SM-3, INX-SM-4, and INX-SM-6 exhibit inhibition of IL-1β production. INX-SM-3 appears to be the most potent of these compounds tested. In contrast, the S stereoisomer at the acetal position, -INX-SM-56-, showed no inhibition.

Experiment 3: Evaluation of the inhibitory effects of steroid payloads INX-SM-9, INX-SM-31, and INX-SM-35 on cytokine production of LPS-stimulated human PBMCs In this experiment, novel glucocorticoid payloads INX-SM-9, The anti-inflammatory efficacy of INX-SM-31 and INX-SM-35 was evaluated. PBMC from two donors were tested. Specifically, Figure 53 shows that INX-SM-9, INX-SM-31, and INX-SM-35 inhibit IL-1β (top) and IL-6 (bottom) production. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.2 nM), with <0.2 nM marked as treatment control on the x-axis on a logarithmic scale. is not plotted, n=2 donors, and a representative donor is shown. Standard deviation plotted from technical duplicates.

As shown by the results in Figure 53, INX-SM-9, INX-SM-35, and INX-SM-31 exhibit dose-dependent inhibition in IL-1β production. INX-SM-31 appears to be the least potent of these compounds tested.

Experiment 4: Evaluation of the inhibitory effect of an exemplary steroid payload INX-SM-32 on cytokine production of LPS-stimulated human PBMCs In this experiment, the anti-inflammatory efficacy of the novel glucocorticoid payload INX-SM-32 was evaluated. The experiment was repeated with PBMC from a second donor. Specifically, the data in Figure 54 shows that INX-SM-32 inhibits the production of IL-1β (top) and IL-6 (bottom). Cytokine levels were measured over 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (500-1 nM), with no treatment control plotted on the x-axis on a logarithmic scale for <1 nM. , n=2 donors. Representative donors are shown. Standard deviation plotted from technical duplicates.

As shown from the data in Figure 54, INX-SM-32 exhibits dose-dependent inhibition in IL-1β and IL-6 production.

Experiment 5: Evaluation of the inhibitory effect of exemplary steroid payloads INX-SM-10 and INX-SM-33 on cytokine production of LPS-stimulated human PBMCs. -33 was evaluated for anti-inflammatory efficacy. PBMC from one donor were tested. Figure 55 shows that INX-SM-10 induces potent inhibition in IL-1β (top) and IL-6 (bottom) production. INX-SM-33 demonstrated slight inhibition of cytokine production. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.5 nM), with <0.5 nM marked as treatment control on the x-axis on a logarithmic scale. is not plotted, n=1 donor, and standard deviations are plotted from technical duplicates.

More specifically, the data in Figure 55 show that INX-SM-10 and INX-SM-33 induce a dose-dependent inhibition of IL-1β production, and that INX-SM-33 It appears to be the least potent of these compounds.

Experiment 6: Inhibitory effects of exemplary steroid payloads INX-SM-2, INX-SM-7, INX-SM-13, INX-SM-24, and INX-SM-74 on cytokine production of LPS-stimulated human PBMCs. Evaluation In this experiment in Figure 56, the anti-inflammatory efficacy of the novel glucocorticoid payloads INX-SM-2 and INX-SM-7 was evaluated. In addition, INX-SM-13 (halogenated at C9), INX-SM-24 (halogenated at C6 and C9), and INX-SM-74 (S stereoisomer of INX-SM-24) INX-SM-3 (non-halogenated) was evaluated to establish the effect of steroid ring halogenation on these compounds. In this experiment, PBMCs from a single donor were used.

Specifically, the data in Figure 56 demonstrate dose-dependent inhibition of IL-1β production by INX-SM-2 and INX-SM-7. Specifically, in the figure, mean cytokine levels were measured over 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.16 nM), and <0.16 nM. No treatment control was plotted on the log scale x-axis, n=1, and standard deviations were plotted from technical duplicates.

Additionally, as shown in Figure 57, evaluation of the effect of halogenation on the potency of INX-SM-3 shows that fluorination at both the C6 and C9 positions of INX-SM-24 -3 was demonstrated to result in increased potency. However, fluorination alone at the C9 position (INX-SM-13) did not result in increased potency over the non-fluorinated payload (INX-SM-3). Notably, the S stereoisomer of INX-SM-24 also showed dose-dependent efficacy. This is in contrast to several non-fluorinated S stereoisomers tested (INX-SM-53, INX-SM-54, and INX-SM-56), which did not show similar efficacy in similar in vitro studies. different.

The data in Figure 57 shows that halogenation at both C6 and C9, but not C9 alone, provides increased efficacy. Mean cytokine levels were measured over 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.16 nM), with <0.16 nM treated on the x-axis on a logarithmic scale. No controls were plotted, n=1, and standard deviations were plotted from technical duplicates.

Experiment 7: Evaluation of the anti-inflammatory effects of different ADCs, including those conjugated with antibodies specific for other immune cell targets, including an exemplary inventive linker payload (INX P linker payload). We evaluated the anti-inflammatory effects of ADCs containing other immune cell targets (two different immune cell antigens other than VISTA) and specific antibodies of ADCs containing a linker payload of the present invention (INX P linker payload), and specifically , antibodies were directed against target B (INX1400P) and target A (INX1302P) to detect their effects on LPS-stimulated human PBMCs. As mentioned above, target A and target B are distinct antigens from each other and from VISTA, and both are highly expressed by certain immune cell types.

In this study, we evaluated the anti-inflammatory effects of antibodies conjugated with the INX P linker payload, but directed against other cell surface targets, anti-target B (INX1400P) and anti-target A (INX1302P). Unconjugated anti-target A antibody (INX1302) was added as a control for potential antibody effects. PBMC from one donor were tested.

As shown in Figure 58, all three INXP conjugates were able to deliver active free payload and retain the potency observed with the anti-VISTA INXP conjugate. Figure 58 shows that INX P-conjugated antibodies directed against target B and target A (two different immune cell-specific antigens other than VISTA) also elicit potent anti-inflammatory effects. Mean cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of glucocorticoid conjugate (conjugate payload from 8,000 to 0.26 nM). n=1, mean technical overlap.

In addition, two free payloads, INX-SM-43 and INX-SM-44, were evaluated for anti-inflammatory effects. Both payloads were able to have some anti-inflammatory effect as shown in Figure 59 (INX-SM-43) and Figure 60 (INX-SM-44).

Specifically, the experiment in Figure 59 shows that INX-SM-43 induces moderate inhibition of huIL1-β. Mean cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of glucocorticoid payload (100-0.032 nM). n=1, mean technical overlap. The experiment in Figure 60 also shows that INX-SM-44 induces moderate inhibition of huIL1-β. Mean cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of glucocorticoid payload (1000-0.32 nM). n=1, mean technical overlap.

Experiment 8: Evaluation of the anti-inflammatory efficacy of exemplary glucocorticoid payloads INX-SM-25, INX-SM-45, and INX-SM-46 vs. INX-SM-3) In this study, the novel glucocorticoid payload INX- The anti-inflammatory efficacy of SM-25, INX-SM-45, and INX-SM-46 versus INX-SM-3 was evaluated. PBMC from one donor were tested. Specifically, Figure 61 shows that INX-SM-25 and INX-SM-3 exhibit potent inhibition in IL-1β production. INX-SM-45 and INX-SM-46 demonstrated less inhibition of cytokine production. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.5 nM), with <0.5 nM marked as treatment control on the x-axis on a logarithmic scale. is not plotted, n=1 donor, and the mean of technical duplicates is plotted.

As shown in the experiments in Figure 61, all free payloads tested showed dose-dependent inhibition in IL-1β production, with INX-SM-45 and INX-SM-46 showing a dose-dependent inhibition in IL-1β production; Compared to INX-SM-3, it has clearly reduced efficacy.

Experiment 9: Evaluation of anti-inflammatory efficacy of novel glucocorticoid payloads in fully conjugated form (INX231J, INX231P, INX231V)
In this study, data shown in Figure 62, the anti-inflammatory efficacy of a novel glucocorticoid payload in fully conjugated form was evaluated. The previously reported linker/payload INX J (INX-J2) was compared to the new linker payload INX P containing the INX-SM-3 payload and INX V containing the INX-SM-32 payload. All linker payloads were conjugated to the anti-VISTA antibody INX231 as INX231J, INX231P, and INX231V with a DAR of approximately 8. Notably, all payloads (INX-SM-3), (INX-SM-32), and (INX-SM-3 and INX-J2) showed significantly lower efficiencies compared to budesonide in in vitro stimulated PBMC assays. It had similar efficacy.

Conjugates were added to single donor PBMCs as a dose response of payload concentration and incubated for 4 hours. This delay caused payload processing to take longer. After 4 hours, LPS was added and samples were incubated for 24 hours before evaluating IL-1b and IL-6. As shown in Figure 62, INX231V dramatically enhanced potency over INX231P and INX231J. INX231P showed some improvement in efficacy over INX231J (literature comparison). This finding was unexpected since the same linker (gly/glu) was used and all free payloads had similar potency compared to budesonide.

This finding was unexpected since the same linker (gly/glu) was used and all free payloads had similar potency compared to budesonide. In particular, Figure 62 shows a dramatic increase in the potency of INX231V over INX231P and INX231J. A. for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM). huIL-1βB. Mean cytokine levels of huIL-6 were measured at 24 hours, <0.16 nM, no treatment control plotted on the x-axis on a logarithmic scale, n=1, and standard deviations were plotted from technical duplicates. Values above the ULOQ (12,500 pg/mL for IL-1b and 150,000 pg/mL for IL-6) were plotted as inserted values.

Experiment 10: Evaluation of anti-inflammatory efficacy of novel glucocorticoid payloads in fully conjugated form (INX231J, INX231P, INX234P, INX231V, INX234A4, INX231S, INX234T, INX234A3, INX201J, INX201L, INX234A11, INX2 34V, INX234A5)
In this study, data shown in Figure 63, the anti-inflammatory efficacy of a novel glucocorticoid payload in fully conjugated form was evaluated. Specifically, INX231 conjugated with the previously reported linker/payload, INX J (INX-J2), was used to synthesize INX231P, INX234P, INX231V, INX234A4, INX231S, INX234T, INX234A3, INX201J, INX201L, INX234 A11, Compared with INX234V and INX234A5. PBMC from one donor were tested.

As shown in the experiment in Figure 63, conjugate INX V has a substantial potency enhancement over conjugates INX P and INX J when V is conjugated to the same anti-target antibody. Conjugate INX P has slight enhancement over conjugate INX J. This is consistent with the accumulation data in human PBMCs disclosed herein showing 235 times more released payload for INX231V compared to INX231P and more than 3 times more released payload for INX231P compared to INX231J. Of note, INX234P and INX231P have similar potency, reflecting similar properties of these antibodies.

The experiment in Figure 64 further shows that INX231V has substantial potency and INX231P has marginal potency compared to INX231J. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted. Specifically, as shown in the experiment in Figure 64, INX231S is more effective than other conjugates, which may be due to the enhanced potency of its released C6/C9 fluorinated payload (INX-SM-24). It has substantial effect on the gate. INX234T (a phosphorylated version of INX P) and INX234A3 (a positively charged linker variant of INX V) also have greater potency than INX231J. Specifically, the experiment in Figure 64 shows the enhanced efficacy of INX231S, INX234T and INX234A3 over INX231J. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted.

Although INX231 and INX234 conjugates have been observed to behave similarly, linker payloads conjugated with INX201 may have enhanced potency in vitro due to more rapid internalization of INX201 antibodies. Indeed, as shown in the experiment of Figure 65, INX201J and IN201L have enhanced potency compared to the INX231J conjugate. Of note, INX234A4 (presumably equivalent to the INX231A4 conjugate form) has enhanced potency over INX231J. In particular, FIG. 65 shows that INX201 can lead to improved early efficacy of the conjugate versus INX231. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted.

Furthermore, as shown in the experiment in Figure 66, the negatively charged linker variant of V, INX A11, utilizes an Asn/gly linker as opposed to the Glu/gly linker of INX V, has similar potency enhancement compared to Of note, INX234A5 also contains spiro[2.2]heptane and exhibits enhanced potency compared to INX231J, suggesting the importance of release and subsequent efficacy. Specifically, the experiment in Figure 66 shows that different analogs of INX V are more potent compared to INX231J. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted.

Furthermore, the efficacy of INX-SM-3, INX-J2, budesonide, dexamethasone, INX-SM-32, and INX-SM-36 was compared. As shown in the experiment in Figure 67, INX-SM-36, INX-SM-32 (top) and INX-SM-3, INX-SM-J2 (bottom) inhibit IL-1β. INX-SM-32 and INX-SM-36 inhibit IL-1β with similar potency as dexamethasone. INX-SM-3 and INX-J2 have similar efficacy to budesonide. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.5 nM), with <0.5 nM marked as treatment control on the x-axis on a logarithmic scale. is not plotted, n=1 donor, and the mean of technical duplicates is plotted.

Experiment 11: Evaluation of the anti-inflammatory efficacy of novel glucocorticoid payloads (INX-SM-3, INX-SM-37, and INX-SM-32) relative to the reported glucocorticoid payload (INX-J2). The anti-inflammatory efficacy of glucocorticoid INX-J2 (a known free glucocorticoid) was evaluated in comparison to the novel glucocorticoids INX-SM-3, INX-SM-37, and INX-SM-32. Direct comparison of the free payloads of the INX J, INX P, and INX V linker payloads (INX-J2, INX-SM-3, and INX-SM-32, respectively) shows that all Shows similar efficacy. The results also suggest that INX-J2 may show a slight potency enhancement over INX-SM-32 and INX-SM-3. This similarity in potency is attributable to the potency observed with the INX V and INX P conjugates (and related analogs) compared to the INX J conjugate, as shown in the experiments in Figures 68 and 64. We demonstrate conclusively that the enhancement is likely due to enhanced accumulation rather than enhanced potency of the released payload. In contrast, INX-SM-37 has weak potency compared to other free payloads.

The data in Figure 68 show that INX-SM-32, INX-J2, and INX-SM-3 have similar inhibition of IL-1β, that INX-SM-37 weakly inhibits IL-1β, and that INX- We show that SM-32, INX-J2, and INX-SM-3 inhibit IL-1β with similar efficacy. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.15 nM), with <0.5 nM marked as treatment control on the x-axis on a logarithmic scale. is not plotted, n=1 donor, and the mean of technical duplicates is plotted.

The experiment in Figure 69 further shows that INX231V is substantially more potent than other INX231/INX234 conjugates. In this experiment, mean cytokine levels of IL-1β were measured over 24 hours in human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM) and <0. At 16 nM no treatment control was plotted on the x-axis on a logarithmic scale, n=1, and the mean of technical duplicates was plotted.

As shown in Figure 70, the phosphorylated (INX231A7, INX231A12) and halogenated forms (INX231A23 and INX231A12) of INX V also exhibit enhanced potency over INX 231J. In particular, the experiments in Figure 70 show that phosphorylated and halogenated analogs of INX V are more potent compared to INX J. Mean cytokine levels of IL-1β were measured over 24 hours on human PBMCs incubated with 1 ng/mL LPS and serial dilutions of conjugated steroid payload (1000-0.16 nM), logarithmic scale for <0.16 nM. No treatment control was plotted on the x-axis, n=1, and the mean of technical duplicates was plotted.

Experiment 12: New glucocorticoid payload (INX-SM-14, INX-SM-15, INX-SM-17, INX-SM-40, INX-SM-34, INX-SM-47, INX-SM-49, and INX-J2) In this study, we evaluated the anti-inflammatory potency of a novel glucocorticoid payload. The previously reported payload INX-J2 was replaced with payloads INX-SM-14, INX-SM-15, INX-SM-17, INX-SM-40, INX-SM-34, INX-SM-47, and INX-SM-49. PBMC from one donor were tested.

As shown in Figure 71, INX-SM-14 and INX-SM-15 have similar ability to inhibit IL-1β and IL-6 as INX-J2. As shown in FIGS. 71, 72, and 73, the other GC payloads, INX-SM-17, INX-SM-34, INX-SM-40, INX-SM-47, and INX-SM-49, Compared to INX-J2, it weakly inhibits IL-1β and IL-6, or only weakly inhibits IL-1β (INX-SM-17).

In particular, the data in Figure 71 show that INX-SM-14, INX-SM-15, and INX-J2 have similar inhibition on IL-1β (top) and IL-6 (bottom). . INX-SM-17 weakly inhibits IL-1β but not IL-6. INX-SM-14, INX-SM-15 and INX-J2 inhibit IL-1β and IL-6 with similar efficacy. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.15 nM), with treatment control on the x-axis of a logarithmic scale at 0.01 nM. Not plotted, n=1 donor, mean of technical duplicates plotted.

Additionally, the data in Figure 72 show that INX-SM-40 and INX-SM-34 weakly inhibit IL-1β (top) and IL-6 (bottom) compared to INX-J2. Cytokine levels were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.15 nM), with treatment control on the x-axis of a logarithmic scale at 0.01 nM. Not plotted, n=1 donor, mean of technical duplicates plotted.

The data in Figure 73 also shows that INX-SM-49 and INX-SM-47 weakly inhibit IL-1β (top) and IL-6 (bottom) compared to INX-J2. Cytokine levels (IL1-β and IL-6) were measured at 24 hours for human PBMCs incubated with 1 ng/mL LPS and serial dilutions of steroid payload (1000-0.15 nM), logarithmic at 0.01 nM. No treatment control was plotted on the x-axis of the scale, n=1 donor, and the mean of technical duplicates was plotted.

Experiment 13: Evaluation of anti-inflammatory efficacy of novel glucocorticoid payloads in fully conjugated form (INX234J, INX201J, INX234A9, INX201V)
In this study, we evaluated the anti-inflammatory efficacy of a novel glucocorticoid linker payload in its fully conjugated form. A previously reported linker payload, INX-J2, was conjugated to INX234 and INX201 and compared to INX A9 conjugated to INX231 and INX V conjugated to INX201. PBMC from one donor were tested. As shown in Figure 74, INX A9 and INX V exhibit enhanced potency over INX J conjugated to the same antibody scaffold. Notably, INX A9 contains a spiro ring system different from that of INX V in the adapter region of the payload, and a conjugated form more similar to INX V than would have been predicted by the free payload. Showing enhanced potency (INX-SM-46 shown in Figures 118A-O). This is likely due to the enhanced accumulation observed with INX V.

In particular, the data in Figure 74 show that INX231A9 and INX201V exhibit enhanced efficacy over INX234J and INX201J in reducing IL-1β production. Cytokine levels were measured at 24 hours for human PBMCs incubated with serial dilutions of anti-VISTA conjugate to 1 ng/mL LPS and conjugate payload concentrations (1000 to 0.15 nM), and 0.1 nM on a logarithmic scale. No treatment control was plotted on the x-axis, n=1 donor, and the mean of technical duplicates was plotted.

Experiment 14: Evaluation of anti-inflammatory efficacy of novel glucocorticoid payloads in fully conjugated form (INX231V, INX231J, INX201A23, INX201V, INX201J, INX201P) (DAR 4 vs. DAR 8)
In this study, the anti-inflammatory efficacy of a novel glucocorticoid payload was evaluated in its fully conjugated form, and in particular the impact of a reduced DAR (approximately 4 vs. approximately 8). Conjugates INX231V (DAR 3.66), INX201A23 (DAR 4.34 and 7.71), INX201V (DAR 3.95 and 7.86), and INX201P (DAR 4.52) were combined with previously reported linkers. / payload, INX J (INX-J2) conjugated with INX201 and INX231, both evaluated at DAR 8.0. PBMC from one donor were tested.

As shown in Figure 75, the INX V conjugate at a reduced DAR (3.66) was more active in reducing IL-1β than the INX J conjugate with the same antibody backbone at a DAR of 8.0. It has high potency. Similarly, INX201A23, which contains a payload similar to INX V, has increased potency with both DAR 7.71 and DAR 4.43 over INX231V DAR 3.66 and INX231J DAR 8.0 due to C6, C9 fluorination. have In particular, the data in Figure 75 show that INX V and INX A23 at equivalent or reduced DAR exhibit enhanced efficacy over INX J in reducing IL-1β production. Cytokine levels were measured at 24 hours on human PBMCs incubated with serial dilutions of anti-VISTA conjugate to 1 ng/mL LPS and conjugate payload concentrations (20 to 0.003 μg/mL) and 0.001 μg/mL. No treatment control was plotted on the x-axis on a logarithmic scale, n=1 donor, and the mean of technical duplicates was plotted.

The experiment in Figure 76 further shows that INX201V at DAR 7.86 or a reduced DAR of 3.95 demonstrates more efficacy than INX201J DAR 8.0. Of note, INX201P (DAR 4.52) has similar efficacy to INX201J (DAR 8.0) despite having a 43% lower DAR than the equivalent INX J conjugate. For both the INX201V and INX201A23 conjugates, the conjugate with a DAR of about 8 had approximately twice the potency of the conjugate with a DAR of about 4. Given the potency profile of these and other novel linker payloads described herein, DAR4 may be sufficient for therapeutic use and may have favorable development characteristics.

More specifically, the data in FIG. 76 shows that INX V conjugates have enhanced potency than INX J conjugates in affecting IL-1β, even with reduced DAR. In experiments, cytokine levels were measured over 24 hours in human PBMCs incubated with serial dilutions of anti-VISTA conjugate to 1 ng/mL LPS and conjugate payload concentrations (20-0.003 μg/mL) and 0. At 0.001 μg/mL, no treatment control was plotted on the x-axis on a logarithmic scale, n=1 donor, and the mean of technical duplicates was plotted.

Conclusion Conclusions on the Novel Glucocorticosteroids The following novel GCs were shown to exhibit varying degrees of dose-dependent steroid potency as free payloads in in vitro assays using LPS-activated human PBMCs:
INX-SM-1, INX-SM-2, INX-SM-3, INX-SM-4, INX-SM-6, INX-SM-7, INX-SM-9, INX-SM-10, INX- SM-13, INX-SM-24, INX-SM-31, INX-SM-32, INX-SM-33, INX-SM-35, INX-SM-36, INX-SM-37, INX-SM- 43, INX-SM-44, INX-SM-45, INX-SM-46, INX-SM-74, INX-SM-14, INX-SM-15, INX-SM-17, INX-SM-34, INX-SM-40, INX-SM-47, INX-SM-49

The lowest potency among the series of R stereoisomers was observed with INX-SM-31, INX-SM-33, and INX-SM-37.

Evaluation of the effect of fluorination on the potency of INX-SM-3 demonstrated that double halogenation at both the C6 and C9 positions of INX-SM-24 resulted in increased potency. However, fluorination alone at the C9 position (INX-SM-13) did not result in increased efficacy against the non-fluorinated payload (INX-SM-3).

Payloads containing the S stereoisomer in the acetal position (INX-SM-53, INX-SM-54, and INX-SM-56) showed no efficacy. An exception to this is INX-SM-74, which is halogenated at both the C9 and C6 positions and has moderate potency, albeit much weaker than the R stereoisomer with the same halogenation. showed that.

Changing the acetal to pyrrolidine (eg, converting INX-SM-32 to INX-SM-37) dramatically reduced the potency of the free payload.

Conclusions regarding the conjugate linker payload: The data show that:
• In these conjugate forms, the majority of the linker payload retains the potency of the free payload.
• Linker payloads with spiro[3.3]heptane (INX V series) had enhanced potency compared to the prior art linker payload (INX J) to levels not predicted by free payload potency. This enhanced efficacy may be due to enhanced release resulting in higher levels of accumulation, as seen in the previous example.
• These linker payloads with enhanced potency include: INX V, INX A11, INX A3, INX A4, INX A5, INX A12, INX A7, INX A23, and INX A9.
The enhanced potency of the conjugate is as reasonably expected given the approximately 50% reduction in payload per antibody, even though the total potency is approximately half that of the corresponding DAR conjugate of approximately 8. Stored at about 4 DAR. Despite the reduced potency, a DAR of 4 may be sufficient for therapeutic applications and may have more desirable development properties than a DAR 8 molecule.
• Another linker payload with spiro[2.5]octane also showed enhanced potency against INX J to a level not predicted by the potency of the free payload INX-SM-46.
• The INX P conjugate had slightly enhanced potency over the INX J conjugate with the same antibody backbone due to enhanced release resulting in higher levels of accumulation. This increase in potency results in an INX P conjugate with a 43% lower DAR that is as potent as an INX J conjugate with the same antibody backbone.
• INX201 conjugates with the same linker payload had increased potency than INX231 conjugates. INX234 and INX231 conjugates with the same linker payload have similar efficacy.
- INX P conjugates directed against other cell surface targets (e.g. anti-target AP, anti-target BP) are also potent in this assay, and because the linker payload is potent. , confirmed that conjugation to specifically anti-VISTA targeting antibodies was not required.

The data further show that:
• The steroid structure can accommodate various alternative shapes, ring sizes, and structures of the C17/C16 acetal while maintaining potency.
• However, only the R isomer at the acetal carbon of non-halogenated steroids is allowed. Of note, the presence of fluorination at both positions C6 and C9 on the steroid ring allows for the potency of the S isomer, although it is much weaker than the corresponding R isomer.
●R isomer: INX-SM-1, INX-SM-2, INX-SM-3, INX-SM-4, INX-SM-6, INX-SM-7, INX-SM-09, INX-SM -10, INX-SM-13, INX-SM-24, INX-SM-31, INX-SM-32, INX-SM-33, INX-SM-35, INX-SM-43, INX-SM-44 , INX-SM-45, INX-SM-46, INX-SM-36
●S isomer: INX-SM-53, INX-SM-54, INX-SM-56, INX-SM-74 (fluorinated with C6/C9)
● Pyrrolidine analog (INX-SM-37) instead of acetal is less potent but still maintains some potency ● INX V with some payloads, especially the INX-SM-32 payload and its analogs The conjugate form of can potentially allow substantial enhancement of efficacy due to enhanced release and exposure of the corresponding free payload.
●When antibodies are held constant, the conjugated form of INX P is slightly more potent than the free payload form of INX J (INX-SM-J2) than that of INX P (INX-SM-3). but with a slight enhancement in potency over the INX J conjugate.

Example 10: Pharmacokinetic Evaluation of Exemplary Anti-VISTA Antibodies This example defines the pharmacokinetics (PK) of various anti-human VISTA antibodies according to the present invention, using BMS (767-IgG1.3, Johnston et al. A study was conducted to compare the same to the pH-sensitive anti-human VISTA from 2019).

The objectives of this experiment were to 1) confirm that the "pH sensitive" antibody described by BMS/Five Prime Therapeutics has a significantly different PK (comparable to hIgG1) compared to the ImmuNext (INX) anti-VISTA antibody; 2) to evaluate the PK of more INX anti-VISTA antibodies (see INX200 and other INX antibody sequences in Figures 8, 10, and 12) and to further evaluate the PK of more other anti-VISTA antibodies; (See other INX antibody sequences in Figures 8, 10, and 12).

These studies were performed in human VISTA knock-in (hVISTA KI) mice that were knocked in with human VISTA cDNA in place of the mouse VISTA gene and express human VISTA at both RNA and protein levels. Experiments were performed in female or male hVISTA KI mice, and in all studies animals received a single dose of antibody at 10 mg/Kg. The amount of antibody in peripheral blood was quantified by ELISA.

Materials and Methods Experimental Design Experiment 1:
hVISTA KI mice were divided into two groups of 10 mice each and treated on day 0 with a single dose of human IgG1 and INX200 at 10 mg/Kg, respectively.

Experiment 2:
hVISTA KI mice were divided into two groups of 10 mice each and treated on day 0 with a single dose of human IgG1 and 767-IgG1.3 at 10 mg/Kg, respectively.

In both Experiment 1 and Experiment 2, 5 mice were incubated at 20 minutes, 4 hours, 24 hours, 48 hours, and then on days 5 and 8 for experiment 1 and days 4 and 7 for experiment 2. Blood was collected from the retroorbital cavity and circulating antibodies were quantified by ELISA. These results are in Figures 77 and 78, respectively.

Experiment 3:
hVISTA KI mice were divided into 4 groups of 15 mice each and treated on day 0 with a single dose of INX231, INX234, INX237, and INX240 at 10 mg/Kg, respectively. Five mice per group were bled retroorbitally at 20 minutes, 4 hours, 24 hours, and then on days 2, 3, 4, 5, 8, 11, 14, and 21. These results are in FIG. 79.

Experiment 4:
hVISTA KI mice were divided into 4 groups of 10 mice each and treated on day 0 with a single dose of INX901, INX904, INX907, and INX908 at 10 mg/Kg, respectively. Seven mice per group were bled retroorbitally at 30 minutes, 4 hours, 24 hours and then on days 2, 3, 4, 7 and 14. These results are in Figure 80.

Experiment 5:
hVISTA KI mice were divided into 5 groups of 4 mice each and treated on day 0 with a single dose of INX201J, INX231J, INX234J, and INX240J at 10 mg/Kg, respectively. On days 3 and 6, mice were bled retroorbitally. These results are shown in FIG.
Test Agent and Dosage INX200 (Aragen, lot number BP-2875-019-6.1) is a humanized anti-human VISTA antibody on a human IgG1/kappa backbone with L234A/L235A silencing mutations in the Fc region.
INX201 (Aragen, lot number BP-3200-019-6) is a humanized anti-human VISTA antibody (same variable domain as INX200) on a human IgG1/kappa backbone with L234A/L235A/E269R/K322A silencing mutations in the Fc region. ).
Human IgG1 (BioXcell ref, lot number 659518N1)
767-IgG1,3 (Aragen, lot number BP-2985-019-6) is a Five Prime Therapeutics and Bristol-Myers Squibb Comp on a human IgG1/kappa backbone with L234A/L235E/G237A silencing mutations in the Fc region. by any This is an anti-human VISTA antibody developed. This antibody was designed to bind at low pH (eg, pH 6), but with minimal binding at physiological pH (pH 7.4).
INX231, INX234, INX237, and INX240 (lot numbers 72928.1.a, 72931.1.a, 72934.1.a, and 73419.1.a, respectively) have L234A/L235A/E269R/K322A in the Fc region. Humanized anti-human VISTA antibody on human IgG1/kappa backbone with silencing mutations.
INX201J, INX231J, INX234J, and INX240J (lot numbers JZ-0556-027, JZ-0556-013-1, JZ-0556-013-2, JZ-0556-013-3) are fully modified with interchain disulfides. INX201, INX231, INX234, INX237, and INX240, respectively, with a drug/antibody ratio (DAR) of 8.0. The linker/payload (J) is based on a patent reported linker/payload consisting of a protease sensitive linker with a budesonide analog payload.
INX901, INX904, INX907, and INX908 are humanized anti-human VISTA antibodies on a native human IgG2/kappa backbone, and the variable domains match INX231, INX234, INX237, and INX200/INX201, respectively.

All antibodies were diluted in PBS and injected intravenously into the tail vein of mice in a volume of 0.2 ml to deliver a dose of 10 mg/Kg.

Mice hVISTA mice were bred at Sage Labs (Boyertown, PA). Mice aged 8-12 weeks were initially transitioned to a quarantine facility for 3 weeks and then transferred to regular facilities. They were allowed to acclimate for 1-2 weeks before starting the experiment.

Bleeding and Preparation Animals were bled no more than once every 24 hours. Each group of mice was divided into two or three subgroups of five mice, which were alternately bled on day 0. Blood was collected on day 0, 20 minutes, 4 hours, 24 hours, 48 hours post-injection, and then on days 5 and 8 for experiment 1 and days 4 and 7 for experiment 2. In the first 24 hour period, some data were excluded based on the registered quality of the intravenous injection. At subsequent time points, only animals that were successfully injected intravenously were bled.

For Experiment 3, mice were bled at 20 minutes, 4 hours, 24 hours, and then on days 2, 3, 4, 5, 8, 11, 14, and 21.

For experiments 4 and 5, mice were bled at 30 minutes, 4 hours, 24 hours, and then on days 2, 3, 4, 7, and 14.

Peripheral blood was collected from the retroorbital cavity using a glass Pasteur pipette that was first rinsed with heparin to prevent clotting. Blood was then centrifuged at 400 rcf for 5 minutes and plasma was collected and stored at -80°C for analysis (see below).

Antibody blood concentration analysis ELISA for detection of human IgG1
First, a 96-well flat bottom plate (Thermo Scientific Nunc Immunol Maxisorp, Cat. No. 442404) was incubated with mouse anti-huIgG Fcγ (Jackson ImmunoResearch, Cat. No. 209-00) at 1 μg/ml in PBS for 1 hour at room temperature (RT). 5-098) Coated with.

Wells were washed three times with PT (PBS with 0.05% Tween 20) and then blocked with PTB (PBS with 0.05% Tween 20 and 1% BSA) for 1 hour at room temperature. Human IgG (Southern Biotech, catalog number 0150-01) was used as a positive control and human IgG1 (BioXcell, catalog number BE0297) was used to generate a standard curve. Wells were washed three times with PT and then plasma samples were incubated for 1 hour at room temperature at up to four different dilutions in PTB (to fit on the standard curve).

After washing three times with PT, mouse anti-human IgG Fcγ conjugated to HRP (Jackson ImmunoResearch, catalog number 209-035-098) was used as the detection reagent at a dilution of 1/2000 and incubated for 1 hour at room temperature. After washing three times, the ELISA reaction was revealed using TMB (Thermo Scientific, Cat. No. 34028) as a colorimetric substrate. After 5-10 minutes at room temperature, the reaction was stopped with 1M H ₂ SO ₄ .

ELISA detection of INX200 (Experiment 1)
First, a 96-well flat bottom plate (same as 4.5.1) was coated with hIX50 (human VISTA ECD, produced at Aragen Bioscience for ImmuNext) at 1 μg/ml in PBS for 1 hour at room temperature.

After washing three times, the wells were blocked with PTB for 1 hour at room temperature. INX908 (produced by Aragen Bioscience for ImmuNext) was used as a positive control and a standard curve was generated using INX200. Wells were washed three times with PT and then plasma samples were incubated for 1 hour at room temperature at up to four different dilutions in PTB (to fit on the standard curve).

After washing three times with PT, mouse anti-human kappa-HRP (Southern Biotech, catalog number 9230-05) was used at 1/2000 as a detection reagent and incubated for 1 hour at room temperature. After washing three times, ELISA reactions were revealed using TMB substrate. After 5 minutes _at room temperature, the reaction was stopped with 1M _H2SO4 .

ELISA detection of 767-IgG1.3 (Experiment 2)
First, a 96-well flat bottom plate (same as above) was coated with mouse anti-huIgG Fcγ (Jackson ImmunoResearch, Cat. No. 209-005-098) at 1 μg/ml in PBS for 1 hour at room temperature.

After washing three times, the wells were blocked with PTB for 1 hour at room temperature. Human IgG (Southern Biotech, catalog number 0150-01) was used as a positive control and 767-IgG1.3 was used to generate a standard curve. Wells were washed three times with PT and then plasma samples were incubated for 1 hour at room temperature at up to four different dilutions in PTB (to fit on the standard curve).

After washing three times with PTB, mouse anti-human IgG Fcγ-HRP (Jackson ImmunoResearch, catalog number 209-035-098) was used at 1/2000 as a detection reagent and incubated for 1 hour at room temperature. After washing three times, ELISA reactions were revealed using TMB substrate according to the manufacturer's instructions. After 5 minutes _at room temperature, the reaction was stopped with 1M _H2SO4 .

Experiment 3 ELISA
First, a 96-well flat bottom plate (same as above) was coated with hIX50 (human VISTA ECD, produced at Aragen Bioscience for ImmuNext) at 1 mg/ml in PBS for 1 hour at room temperature.

After washing three times, the wells were blocked with PTB for 1 hour at room temperature. INX908 (produced by Aragen Bioscience for ImmuNext) was used as a positive control and standard curves were generated using INX231, INX234, INX237, or INX240. Wells were washed three times with PT and then plasma samples were incubated for 1 hour at room temperature at up to four different dilutions in PTB (to fit on the standard curve).

After washing three times with PTB, mouse anti-human IgG Fcγ-HRP (Jackson ImmunoResearch, catalog number 209-035-098) was used at 1/2000 as a detection reagent and incubated for 1 hour at room temperature. After washing three times, ELISA reactions were revealed using TMB substrate according to the manufacturer's instructions. After 5 minutes _at room temperature, the reaction was stopped with 1M _H2SO4 .

Experiment 4 ELISA
First, a 96-well flat bottom plate (same as previous experiment) was coated with hINX50 (human VISTA ECD, produced at Aragen Bioscience for ImmuNext) at 1 mg/ml in PBS for 1 hour at room temperature.

After washing three times, the wells were blocked with PTB for 1 hour at room temperature. INX201 was used as a positive control and standard curves were generated using INX231, INX234, or INX240. Wells were washed three times with PT and then plasma samples were incubated for 1 hour at room temperature at up to four different dilutions in PTB (to fit on the standard curve).

After washing three times with PTB, mouse anti-human IgG Fcγ-HRP (Jackson ImmunoResearch, catalog number 209-035-098) was used at 1/2000 as a detection reagent and incubated for 1 hour at room temperature. After washing three times, ELISA reactions were revealed using TMB substrate according to the manufacturer's instructions. After 5 minutes _at room temperature, the reaction was stopped with 1M _H2SO4 .

Experiment 5 ELISA
First, a 96-well flat bottom plate (same as above) was coated with hIX7 (human VISTA ECD on mouse IgG2 scaffold) at 1 mg/ml in PBS for 1 hour at room temperature.

After washing three times, the wells were blocked with PTB for 1 hour at room temperature. INX901, INX904, INX907, or INX908 were used as positive controls to generate a standard curve. Wells were washed three times with PT and then plasma samples were incubated for 1 hour at room temperature at up to four different dilutions in PTB (to fit on the standard curve).

After washing three times with PTB, mouse anti-human IgG Fcγ-HRP (Jackson ImmunoResearch, catalog number 209-035-098) was used at 1/2000 as a detection reagent and incubated for 1 hour at room temperature. After washing three times, ELISA reactions were revealed using TMB substrate according to the manufacturer's instructions. After 5 minutes _at room temperature, the reaction was stopped with 1M _H2SO4 .

ELISA Assay Calculation The LOQ is calculated by multiplying the lowest point of the standard curve by the lowest dilution factor used to dilute the sample. For example, if the lowest standard point is 0.3 ng/mL and the lowest standard dilution is 1/400, the LOQ is reported in the same units that the sample is reported, so the LOQ is 0.1 ug/mL. be.

The LOD is determined when the sample OD is indistinguishable from the background OD, an OD of approximately 0.01. No concentration is calculated for LOD, but a concentration of 0 or 0.001 ug/mL is assigned for graphing and PK calculation purposes.

Antibody half-life was determined using the PKsolver program with non-compartmental analysis (NCA) after an intravenous bolus.

The results of Experiments 1-5 are in Figures 77-81, respectively.

Figure 77 contains the results of Experiment 1 comparing the PK of INX200 versus human IgG1. Plasma concentrations of antibodies (SD; n=5/group) at annotated time points in hVISTA KI mice are shown.

Figure 78 contains the results of Experiment 2 comparing the PK of 767-IgG1.3 versus human IgG1. Plasma concentrations of antibodies (SD; n=5/group) at annotated time points in hVISTA KI mice are shown.

FIG. 79 includes the results of Experiment 3 comparing the PK of INX231, INX234, INX237, and INX240. Plasma concentrations of antibodies (SD; n=5/group) at annotated time points in hVISTA KI mice are shown. The left graph shows the y-axis and x-axis at Log10, while for the right graph only the y-axis is at Log10.

FIG. 80 includes the results of Experiment 4 comparing the PK of INX901, INX904, INX907, and INX908. Plasma concentrations of antibodies (SD; n=5/group) at annotated time points in hVISTA KI mice are shown.

Figure 81 includes the results of Experiment 5 comparing the PK of INX201J, INX231J, INX234J, and INX240J. Plasma concentrations of antibodies (SD; n=4/group) at annotated time points in hVISTA KI mice are shown.

Data from these experiments are shown below.

Experiment 1 (Figure 77) shows that the PK of anti-human VISTA antibody INX200 cannot be quantified in plasma 24 hours post-dose due to target-mediated pharmacokinetics (TMDD), whereas human IgG1 control shows an extended half-life more typical of IgG.

Experiment 2 (Figure 78) shows that pH-sensitive anti-human VISTA 767-IgG1,3 exhibits similar PK to the human IgG1 control antibody, with limited binding to its VISTA target and TMDD. This suggests that it will not be provided.

The results of Experiment 3 (Figure 79) show that INX231, INX234, INX237, and INX240 are all still detectable after 24 hours, with INX237 having a uniquely increased half-life.

The results of Experiment 4 (Figure 80) show that the incorporation of different IgG backbones into the INX antibodies of the invention did not significantly alter the antibody half-life.

The results of Experiment 5 (Figure 81) show that the addition of GC payload did not appear to further affect the clearance of INX anti-VISTA antibodies at the two time points analyzed.

These results demonstrate that the anti-VISTA antibodies of the present invention and ADCs containing the same have PK values and clearance properties that make them highly suitable for targeted delivery of desired payloads, particularly steroid payloads, to target immune cells. Indicates that it is suitable.

Example 11: Effect of long-term treatment with INX201J versus dexamethasone on corticosterone levels Glucocorticoid hormones are rapidly synthesized and secreted from the adrenal glands in response to stress. Additionally, under basal conditions, glucocorticoids are rhythmically released in both circadian and ultradian (pulsatile) patterns. These rhythms are important not only for the normal function of glucocorticoid target organs but also for the HPA axis response to stress. Many studies have shown that disruption of glucocorticoid rhythms by long-term GC treatment is associated with disease in both humans and rodents. In humans, the most abundant GC is cortisol, and in mice, corticosterone.

Based on the above, the impact of long-term treatment with the exemplary antibody drug conjugate (ADC) INX201J and anti-human VISTA monoclonal antibody conjugated to a glucocorticoid (GC) payload on the HPA axis, particularly basal corticosterone levels. was evaluated. As discussed below, experiments were carried out using a human VISTA knock-in (hVISTA) protein, in which the human VISTA cDNA was knocked in instead of the mouse VISTA gene, and which expressed human VISTA at both the RNA and protein levels, which had the same expression pattern as mouse VISTA. It was carried out at KI). Experiments were performed in female mice that were first acclimatized for one week to a specific handler who subsequently performed all injections and bled to limit stress-induced changes in basal levels of GCs.

Mice were then injected with 10 or 3 mg/Kg INX201J (0.2 or 0.06 mg/Kg payload, respectively) or 2 or 0.2 mg/Kg dexamethasone (Dex). Dex was administered daily for 4 days, while INX201J was administered on days 1, 3, and 4. On day 5, mice were bled and their corticosterone levels were assessed by ELISA.

Materials and Methods Experimental Design Experiments were performed in female hVISTA KI mice. Mice were then injected with 10 or 3 mg/Kg of INX201J (0.2 or 0.06 mg/Kg payload, respectively) on days 1, 3, and 4 or 2 or 0.2 mg/Kg for 4 consecutive days. of dexamethasone (Dex) was injected daily. A control group that received daily PBS injections was included. On day 5, mice were bled and their plasma corticosterone levels were assessed by ELISA.

The rationale for the dosing schedule is based on other studies (see previous examples) that have shown that the ADCs of the invention have a much longer pharmacodynamic range (>96 hours) than Dex (<24 hours). )based on.
Experiment: (8 mice per group)
Group 1: PBS
Group 2: 2mg/Kg Dex
Group 3: 0.2mg/Kg Dex
Group 4: 10mg/Kg INX201J (0.2mg/Kg payload)
Group 5: 3mg/Kg INX201J (0.06mg/Kg payload)

Test drug and dosage Antibody INX201J (Abzena, lot numbers JZ-0556-025-1, JZ-0556-027, JZ-0556-013) is a human antibody with L234A/L235A/E269R/K322A silencing mutations in the Fc region. Based on INX201, a humanized anti-human VISTA antibody on an IgG1/kappa backbone. INX201J is a conjugated antibody with a drug/antibody ratio of 8.0, conjugated through complete modification of interchain disulfides. The linker/payload (J) consists of a protease sensitive linker with a budesonide analog payload.

Antibodies were diluted in PBS and injected intraperitoneally (i.p.) in a volume of 0.2 ml to deliver specific doses.

Dexamethasone Dexamethasone sterile injection from Phoenix, NDC 57319-519-05 was diluted in PBS and administered as described via intraperitoneal injection.

Mice hVISTA mice were bred at the Center for Comparative Medicine and Research at Dartmouth. All experiments were performed on female mice enrolled between 9 and 15 weeks of age.

Blood Sampling and Preparation Peripheral blood was collected from the retroorbital cavity using a glass Pasteur pipette that was first rinsed with heparin to prevent clotting. Blood was then centrifuged at 550 rcf for 5 minutes and plasma was collected and stored at -80°C prior to corticosterone analysis.

Corticosterone ELISA
ELISA was performed using the Arbor Assay (Catalog Number K014-H5) Corticosterone 5-Pack ELISA Kit according to the manufacturer's included protocol.

Results INX201J has a limited effect on corticosterone levels. As shown in the experiment in Figure 82, adaptation of mice resulted in two animals with one showing very high and the other very low corticosterone levels. resulted in relatively consistent levels of corticosterone in the control group. Figure 82 shows uncorrected data on the left and corrected data on the right (without the two outliers in the control group), showing changes in plasma corticosterone levels. (SEM, one-way ANOVA, excluding PBS control group in right graph with n=6, n=8). As shown, 2 mg/Kg of Dex dramatically reduced basal corticosterone levels, while 0.2 mg/Kg had only a limited but significant effect. In contrast, INX201J had no effect at a payload dose of 0.06 mg/Kg, while a limited reduction was observed at a payload dose of 0.2 mg/Kg.

Conclusion The data show that 2 mg/Kg of Dex dramatically reduced basal levels of corticosterone, whereas at 0.2 mg/Kg the reduction was more limited but still highly significant. (P<0.001). In contrast, INX201J at a payload of 0.2 mg/Kg, which is therapeutically equivalent to 2 mg/Kg Dex, had a more limited effect (ns or P<0.5). At 0.06 mg/Kg, there was no effect on corticosterone levels. (These doses were chosen because INX201J with a payload of 0.2 mg/Kg had similar efficacy as Dex at 2 mg/Kg, as shown in the previous example).

Example 12: Effect of ADCs on Antigen-Specific Responses Glucocorticoids (GCs) are known to have a profound effect on primary immune responses and can significantly influence IgG responses to vaccines. Therefore, a vaccine model was used to evaluate the functionality of the subject antibody drug conjugates (ADCs) in disrupting antigen-specific responses. As discussed in detail below, a murine CD40 agonist antibody (FGK4.5), the OVA peptide SIINFEKL as a model antigen (Ag), and a potent CD8 T cell driving Ag that can be measured using the tetramer technique A standard immunization protocol was used in combination with the TLR agonist Poly(I:C) to drive the response. An additional advantage is that this model also made it possible to evaluate the pharmacodynamic range of ADCs by treating for up to one week before vaccination.

As discussed in detail below, we conducted three studies in which the human VISTA cDNA was knocked in instead of the mouse VISTA gene and expressed human VISTA at both the RNA and protein levels with the same expression pattern as mouse VISTA. was carried out in human VISTA knock-in (hVISTA KI) mice. Briefly, these mice were injected with ADC for up to 7 days prior to vaccination. Dexamethasone (Dex) was used as a positive GC control. Immune responses in peripheral blood were measured on day 6 post-immunization at the peak of anti-Ag responses.

Materials and Methods Experimental Design All four experiments were performed in female mice with 5 mice per group.

Experiment 1: Effect of Dex on Ag-specific responses when administered 2 hours before vaccination. To confirm the effect of Dex on Ag-specific responses when administered 2 hours before vaccination, Fig. The experiments described in 83 were performed in C57Bl/6 mice.
Group 1: PBS
Group 2: 2mg/Kg Dex
Group 3: 0.2mg/Kg Dex

Mice were administered 2 or 0.2 mg/Kg of Dex or PBS intraperitoneally. Two hours later, they received a vaccine cocktail injected intraperitoneally. All these animals were then bled 6 days later and the number of Ag-specific CD8 T cells was quantified.

Experiment 2: Effect of ADC INX201J on Ag-specific responses when administered at different time points before vaccination To assess the effect of ADC INX201J on Ag-specific responses when administered at different time points before vaccination This experiment in Figure 84 was conducted in hVISTA KI mice.
Group 1: PBS
Group 2: 2mg/Kg Dex
Group 3: 0.2mg/Kg Dex
Group 4: INX201J at 10 mg/Kg - Day 1 Group 5: INX201J at 10 mg/Kg - Day 2 Group 6: INX201J at 10 mg/Kg - Day 4

Mice in groups 1-3 were administered intraperitoneally 2 hours before immunization. Mice in groups 4-6 were dosed as indicated 1, 2, or 4 days prior to vaccination. All mice were immunized on day 0. All these animals were then bled 6 days later and the number of Ag-specific CD8 T cells was quantified.

Experiment 3
This experiment in Figure 85 was conducted in hVISTA KI mice to evaluate the effect of multiple ADCs on Ag-specific responses when administered at different time points before vaccination.
Group 1: PBS - 2 hours before vaccine Group 2: 2 mg/Kg Dex - 2 hours before vaccine Group 3: 0.2 mg/Kg Dex - 2 hours before vaccine Group 4: 2 mg/Kg Dex-7 Day Group 5: INX201J at 10mg/Kg - Day 1 Group 6: INX201J at 10mg/Kg - Day 7 Group 7: INX231J at 10mg/Kg - Day 7 Group 8: INX234J at 10mg/Kg - Day 7 Group 9: 10mg/Kg INX240J - Day 7

Mice in groups 1-3 were administered intraperitoneally 2 hours before immunization. Mice in groups 4-9 were dosed as indicated 1 or 7 days prior to vaccination. All mice were immunized on day 0. All these animals were then bled 6 days later and the number of Ag-specific CD8 T cells was quantified.

Experiment 4: Effect of multiple ADCs conjugated to GC payload (P) on Ag-specific responses. To evaluate the effects of multiple ADCs, this experiment in Figure 86 was conducted in hVISTA KI mice.
Group 1: PBS - 2 hours before vaccine Group 2: 2 mg/Kg Dex - 2 hours before vaccine Group 3: 10 mg/Kg INX201J - day 1 Group 4: 10 mg/Kg INX201J - day 7 Group 5 : 10mg/Kg INX231P - Day 7 Group 6: 10mg/Kg INX234P - Day 7 Group 7: 10mg/Kg INX240P - Day 7

Mice in groups 1-2 were administered intraperitoneally 2 hours before immunization. Mice in groups 3-7 were dosed as indicated 1 or 7 days prior to vaccination. All mice were immunized on day 0. All these animals were then bled 6 days later and the number of Ag-specific CD8 T cells was quantified.

Test Agents and Dosage Antibodies INX201, INX231, INX234, and INX240 (lot numbers 72928.1.a, 72931.1.a, and 73419.1.a, respectively) were used in these experiments, all of which , a humanized anti-human VISTA antibody on a human IgG1/kappa backbone with L234A/L235A/E269R/K322A silencing mutations in the Fc region.

INX201J, INX231J, INX234J, and INX240J (lot numbers JZ-0556-027, JZ-0556-013-1, JZ-0556-013-2, JZ-0556-013-3) are fully modified with interchain disulfides. INX201, INX231, INX234, and INX240, respectively, with a drug/antibody ratio (DAR) of 8.0. The linker/payload (J) consists of a protease sensitive linker with a budesonide analog payload.

INX201P, INX231P, INX234P, and INX240P (lot numbers JZ-0556-0271, JZ-0556-017-1, JZ-0556-017-2, JZ-0556-017-3) are fully modified with interchain disulfides. INX201, INX231, INX234, and INX240, respectively, with a drug/antibody ratio (DAR) of 8.0. The linker/payload (P) consists of a protease sensitive linker with a budesonide analog payload.

Each of these antibodies was diluted in PBS and injected intraperitoneally (i.p.) in a volume of 0.2 ml to deliver specific doses.

Dexamethasone Dexamethasone sterile injection from Phoenix, NDC 57319-519-05 was diluted in PBS and administered as described via intraperitoneal injection.

Vaccine Cocktail A standard antibody/peptide/poly(I:C) immunization protocol using 50 μg mouse CD40 agonist antibody (clone FGK4.5) + 50 μg SIINFEKL peptide + 50 μg poly(I:C) per mouse. used. The vaccine is diluted in PBS and injected intraperitoneally in a final volume of 200 μl.

Mice hVISTA mice were bred at the Center for Comparative Medicine and Research at Dartmouth. All experiments were performed on female mice enrolled between 9 and 15 weeks of age. C57Bl/6 mice were purchased from Jackson Laboratories.

Blood Sampling and Immunostaining Peripheral blood was collected from the retroorbital cavity using a glass Pasteur pipette that was first rinsed with heparin to prevent clotting. A single wash protocol was used that allows for absolute blood cell counts.

10 μl of antibody cocktail (see below) was added directly to 50 or 100 μl of blood. After 30 minutes of incubation at room temperature (RT), 600 μl of BD FACS lysis buffer was added to the samples. After incubation for 30 minutes at room temperature, samples were spun at 550 rcf for 5 minutes, washed once in PBS, and resuspended in a fixed volume of PBS. The entire sample was run on a MacsQuant flow cytometer to obtain absolute cell counts.

Antibody panel:
The following antibodies were diluted in PBS.
-CD11a-FITC (BioLegend, clone 2D7, 0.5mg/ml) 1:200
-H-2kb-OVA-PE tetramer (MBL iTag MHC tetramer, catalog number T03000, lot number T1603004), (10 μl/sample)
-CD8-Alexa647 (clone KT15, MBL number D271-A64, 1 mg/ml) (1:800).
-Mouse Fc block (1:200)
Gating strategy:
Gating on FSC versus SSC-lymphocyte populations.
FSC-H vs. FSC-A- Singlet population Gating on CD8+ T cells CD8+→CD11a+ vs. Ova-tet+

Results Experiment 1
As described above, to confirm the effect of Dex on Ag-specific responses when administered 2 hours before vaccination, the experiment of Figure 83 was conducted and performed in C57Bl/6 mice. 2 mg/Kg of Dex dramatically reduced the number of Ag-specific CD8 T cells (OVA tet), a clear reduction when also observed at 0.2 mg/Kg. Specifically, Figure 83 shows the number of Ag-specific CD8 T cells from peripheral blood 6 days after immunization. (SEM, one-way ANOVA, n=5).

Experiment 2
As shown in Figure 84, INX201J at 0.2 mg/Kg of GC payload when administered 24, 48, or 96 hours prior to immunization suppressed Ag-specific (Ova Tet+CD8 T cell) responses. shows similar efficacy in reducing Dex at 2 mg/Kg administered 2 hours before immunization. In this experiment, blood from two naive mice was added at the end to provide a baseline control. More specifically, Figure 86 shows Ag-specific CD8 T cell counts from peripheral blood 6 days post-immunization. The left graph in Figure 84 shows the PBS control group including all samples, and the right graph shows the PBS control group with one outlier removed (SEM, one-way ANOVA, n=5 excluding naive , one sample was excluded in the group containing Dex at 0.2 mg/Kg as an immunization failure).

Experiment 3
Four different ADCs were evaluated in the experiment of FIG. As shown therein, all tested ADCs were effective in reducing Ag-specific (Ova Tet+CD8 T cell) responses when administered 1 or 7 days prior to immunization with a GC payload of 0.2 mg/Kg. It showed remarkable effectiveness. Further evidence suggests that Dex lost efficacy at 0.2 mg/Kg but remained effective when administered at 2 mg/Kg or 7 days prior to immunization. I understand. More specifically, the experiment in Figure 85 shows Ag-specific CD8 T cell numbers from peripheral blood 6 days post-immunization. In this experiment, several samples had to be excluded due to technical issues during processing. PBS group n=3, Dex at 2 mg/Kg n=2, Dex at 0.2 mg/Kg n=3, INX201J D-1 n=5, INX201J D-7 n=2, INX231J D-7 n=3, INX234J D-7 n=5, INX240J D-7 n=4 (SEM, one-way ANOVA, D=day).

Experiment 4
In this experiment, included in Figure 86, four ADCs, each conjugated with a different GC payload (P), were evaluated. As shown therein, a significant decrease in Ag-specific (Ova Tet ⁺ CD8 T cell) responses was observed with INX201P, INX231P, and INX234P when administered 1 or 7 days before immunization, which The effect was comparable to that of Dex administered on day 0 at 2 mg/Kg. As shown there, only INX240P showed little efficacy. More specifically, the data in Figure 86 shows the number of Ag-specific CD8 T cells from peripheral blood 6 days after immunization and, for technical reasons, two samples in PBS, INX231P and INX234P groups. For all other groups, n=5 (SEM, one-way ANOVA).

Conclusion As mentioned above, the data from Experiments 1-4 show the following.
(i) Experiment 1 shows that Dex administered 2 hours before immunization at both 2 and 0.2 mg/Kg efficiently reduced Ag-specific responses.
(ii) Experiment 2 demonstrated that an exemplary ADC conjugate according to the present invention, INX201J, when administered 24 hours, 48 hours, or 96 hours before immunization with GC payload of 0.2 mg/Kg is Ag-specific. Dex at 2 mg/Kg administered 2 hours before immunization is shown to be as effective in reducing clinical responses as Dex administered 2 hours before immunization.
(iii) Experiment 3 showed significant efficacy of four exemplary ADCs in reducing Ag-specific responses when administered with GC payload of 0.2 mg/Kg, 1 or 7 days prior to immunization. Show that. In contrast, Dex lost efficacy at 0.2 mg/Kg but showed efficacy when administered at 2 mg/Kg or 7 days before immunization.
(iv) Experiment 4 shows four exemplary ADCs each conjugated with a different GC payload. Also, with the exception of INX240P, a significant reduction in Ag-specific responses was observed with all ADCs tested when administered 1 or 7 days before immunization.

Overall, these data indicate that:
(i) Exemplary ADCs according to the invention administered at GC payload of 0.2 mg/Kg are equally effective in reducing Ag-specific responses as Dex administered at 2 mg/Kg.
(ii) Both J and P GC payloads have equal efficacy.
(iii) Dex loses efficacy when injected 7 days before immunization, but different ADCs still have significant potency in controlling the development of Ag-specific responses.

Example 13: Efficacy of anti-VISTA antibody drug conjugates in the OVA-asthma mouse model Asthma is clinically induced by airway hyperresponsiveness, inflammatory cell infiltration in the bronchoalveolar lavage fluid (BALF) and bronchial walls, and airway structural changes. It is a complex, well-characterized inflammatory disease. Inhaled glucocorticoids (GCs) are considered the standard treatment for most asthma types. Based on that, a study was conducted to evaluate the therapeutic efficacy of the exemplary antibody drug conjugate (ADC) INX201J in a mouse model of allergic asthma.

Briefly, mice were treated with two injections of ovalbumin (OVA) emulsified in aluminum hydroxide, one week apart, as discussed in detail below and illustrated in the figures referenced in this example. sensitized with One or two weeks later (parts 1 and 2 of the experiment), mice were challenged with daily exposure via inhalation to OVA for 5 consecutive days. Treatment consisted of three doses of 10 mg/Kg INX201J (or 0.2 mg/Kg payload) or 2 mg/Kg dexamethasone (Dex) daily during OVA exposure. Analysis was performed 24 hours after the last challenge.

These experiments were carried out using a human VISTA knock-in (hVISTA KI) that substituted the human VISTA cDNA for the mouse VISTA gene and expressed human VISTA at both RNA and protein levels with the same expression pattern as mouse VISTA or C57Bl/6 mice. ) was performed again in mice. The purpose of these studies was to evaluate the therapeutic efficacy of the ADC, INX201J, compared to free dexamethasone (Dex) in a mouse model of OVA asthma.

To assess the level of efficacy of the ADC, the number of inflammatory cells recruited to the lungs and cytokine production in the BAL were measured by flow cytometry. Systemic responses were assessed by ELISA and production of OVA-specific IgG and IgE was quantified. Finally, a blinded analysis of H&E stained lung sections was performed to obtain a disease score.

As discussed in detail below, these experiments were performed using two different time points of OVA challenge, as we evaluated two different protocols described in the literature in parallel, which can be considered internal replicates. did.

Materials and Methods Experimental Design The experiment consisted of the following groups with 10 female mice per group. Groups 1-3 and 5, 6 are C57Bl/6, and mice in groups 4 and 7 are human VISTA KI mice. All mice in groups 2-7 were sensitized to OVA and challenged with OVA.
Group 1: Naive Group 2: OVA alum-inhalation 14-18 days Group 3: OVA alum-inhalation 14-18 days-2mg/Kg Dex
Group 4: OVA alum-inhalation 14-18 days-10mg/Kg INX201J
Group 5: OVA alum - inhalation 21-25 days Group 6: OVA alum - inhalation 21-25 days - 2mg/Kg Dex
Group 7: OVA alum-inhalation 21-25 days-10mg/Kg INX201J

All mice in groups 2-7 were sensitized with 10 μg/mouse ovalbumin emulsified with aluminum hydroxide.

- Part 1 of the experiment:
Five mice in group 1 (naive) and all animals in groups 2-4 were subjected to OVA inhalation (3% OVA in PBS) for 30 minutes for 5 consecutive days from day 14 to day 18. . 2 mg/Kg Dex was injected intraperitoneally daily from day 14 to day 18. 10 mg/Kg INX201J was administered intraperitoneally on days 13, 15, and 17. Treated animals were sacrificed on day 19.

- Part 2 of the experiment:
Five mice in group 1 and all animals in groups 5-7 were subjected to OVA inhalation (1% OVA in PBS) for 30 minutes for 5 consecutive days from day 21 to day 25. 2 mg/Kg Dex was injected intraperitoneally daily from day 21 to day 25. 10 mg/Kg INX201J was administered intraperitoneally on days 20, 22, and 24. Treated animals were sacrificed on day 25.

The experimental design and analysis are described in the literature (Gueders et al., “Mouse models of asthma: a comparison between C57BL/6 and BALB/C strains regulating bronchial responses “Eness, Inflammation, and Cytokine Production”, Inflamm. Res. (2009) 58:845 -854, Yu et al, “Establishment of different experimental asthma models in mice”, Experimental and Therapeutic Medicine 15:2492 -2498, 2018).

Test Agent and Dosage Antibody INX201J (Abzena, lot numbers JZ-0556-025-1, JZ-0556-027, JZ-0556-013). INX201 is a humanized anti-human VISTA antibody on a human IgG1/kappa backbone with L234A/L235A/E269R/K322A silencing mutations in the Fc region. INX201J is a conjugated antibody with a drug/antibody ratio of 8.0, conjugated through complete modification of interchain disulfides. The linker/payload (J) consists of a protease sensitive linker with a budesonide analog payload. INX201J was diluted in PBS and injected intraperitoneally (i.p.) in a volume of 0.2 ml to deliver specific doses.

Dexamethasone Dexamethasone sterile injection from Phoenix, NDC 57319-519-05 was diluted in PBS and administered as described via intraperitoneal injection.

Ovalbumin Ovalbumin (or albumin from chicken egg white) was purchased from Sigma (A5503) and resuspended in PBS. Administered intraperitoneally or via nebulizer.

Mice hVISTA KI mice were bred at the Center for Comparative Medicine and Research at Dartmouth. All experiments were performed on female mice enrolled at 15 weeks of age. C57Bl/6 mice were purchased from Jackson Laboratories.

OVA Inhalation OVA was delivered via nebulizer using the Kent Scientific (AG-ALSM-0530LG) nebulizer delivery system.

Blood Sampling Peripheral blood was collected from the retroorbital cavity using a glass Pasteur pipette that was first rinsed with heparin to prevent clotting. Blood was then centrifuged at 550 rcf for 5 minutes and 75 μl of plasma was collected and stored at −80° C. before cytokine analysis. Blood cells were resuspended in 75 μl of PBS and processed for immunostaining.

Bronchoalveolar lavage Mice were sacrificed by _CO2 inhalation and bronchoalveolar lavage was performed immediately using 5 x 1 ml of PBS-EDTA (0.5 mM). Cells were collected by gentle manual aspiration. The volume was recorded. Samples with collection volumes less than 4 ml were excluded. After centrifugation at 550 rcf for 5 min, the supernatant was collected and frozen at -80°C for protein evaluation. Cells were resuspended in PBS and processed for immunostaining.

BAL immunostaining BAL cell samples were divided into two and stained with two different antibody panels for lymphocytes and myeloid cells (see Tables 2 and 3). After 30 minutes at 4°C, samples were washed once and resuspended in a fixed volume. Fixed deposits were analyzed on a MacsQuant flow cytometer to obtain comparable cell counts.

Whole Blood Immunostaining A single wash protocol was used that allowed for absolute blood cell counts. 10 μl of antibody cocktail (see below) was added directly to 100 μl of blood. After 30 minutes of incubation at room temperature (RT), 600 μl of BD FACS lysis buffer was added to the samples. After incubation for 30 minutes at room temperature, samples were spun at 550 rcf for 5 minutes, washed once in PBS, and resuspended in a fixed volume of PBS. The entire sample was run on a MacsQuant flow cytometer to obtain absolute cell counts.

Different antibody panels were used on lymphocytes and myeloid cells as shown in Tables 4 and 5.

ELISA
ELISA for IgG, OVA-specific IgG, IgE, OVA-specific IgE
ELISA for mouse IgG1
First, a 96-well flat bottom plate (Thermo Scientific Nunc Immunol Maxisorp, Cat. No. 442404) was coated with goat anti-mouse IgG1 (Southern Biotech, Cat. No. 1070-01) at 1 μg/ml in PBS for 1 h at room temperature (RT). did. Wells were washed three times with PT (PBS with 0.05% Tween 20) and then blocked with PTB (PBS with 0.05% Tween 20 and 1% BSA) for 1 hour at room temperature. A standard curve was generated using mouse IgG1 anti-ovalbumin (Biolegend, Cat. No. 520502). Wells were washed three times with PT and then plasma samples were incubated for 1 hour at room temperature at up to four different dilutions in PTB (to fit on the standard curve).

After washing three times with PT, goat anti-mouse IgG1-HRP (Southern Biotech, Cat. No. 1070-05) was used as a detection reagent at 1/20,000 and incubated for 1 hour at room temperature. After washing three times, ELISA reactions were revealed using TMB substrate according to the manufacturer's instructions. After 5-10 minutes at room temperature, the reaction was stopped with 1M H ₂ SO ₄ .

ELISA for mouse IgG1 anti-ovalbumin
First, a 96-well flat bottom plate (Thermo Scientific Nunc Immunol Maxisorp, Cat. No. 442404) was coated with ovalbumin (Sigma, Cat. No. 1070-01) at 95 μg/ml in PBS for 1 hour at room temperature (RT). Wells were washed three times with PT and then blocked with PTB for 1 hour at room temperature. A standard curve was generated using mouse IgG1 anti-ovalbumin (Biolegend, Cat. No. 520502). Wells were washed three times with PT and then plasma samples were incubated for 1 hour at room temperature at up to four different dilutions in PTB (to fit on the standard curve).

After washing three times with PT, goat anti-mouse IgG1-HRP (Southern Biotech, Cat. No. 1070-05) was used as a detection reagent at 1/20,000 and incubated for 1 hour at room temperature. After washing three times, ELISA reactions were revealed using TMB substrate according to the manufacturer's instructions. After 5-10 minutes at room temperature, the reaction was again quenched with 1M H ₂ SO ₄ .

ELISA for mouse IgE
First, a 96-well flat bottom plate (Thermo Scientific Nunc Immunol Maxisorp, Cat. No. 442404) was coated with goat anti-mouse IgE (Southern Biotech, Cat. No. 1110-01) at 1 μg/ml in PBS for 1 h at room temperature (RT). did. Wells were washed three times with PT and then blocked with PTB for 1 hour at room temperature. A standard curve was generated using mouse IgE anti-ovalbumin (BioRad, catalog number MCA2259). Wells were washed three times with PT and then plasma samples were incubated for 1 hour at room temperature at up to four different dilutions in PTB (to fit on the standard curve).

After washing three times with PT, goat anti-mouse IgE-HRP (Southern Biotech, Cat. No. 1110-05) was used as a detection reagent at 1/2000 and incubated for 1 hour at room temperature. After washing three times, ELISA reactions were revealed using TMB substrate according to the manufacturer's instructions. After 5-10 minutes at room temperature, the reaction was stopped with 1M H ₂ SO ₄ .

ELISA for mouse IgE anti-ovalbumin
First, a 96-well flat bottom plate (Thermo Scientific Nunc Immunol Maxisorp, Cat. No. 442404) was coated with ovalbumin (Sigma, Cat. No. 1070-01) at 95 μg/ml in PBS for 1 hour at room temperature (RT). Wells were washed three times with PT and then blocked with PTB for 1 hour at room temperature. A standard curve was generated using mouse IgG1 anti-ovalbumin (BioRad, catalog number MCA2259). Wells were washed three times with PT and then plasma samples were incubated for 1 hour at room temperature at up to four different dilutions in PTB (to fit on the standard curve).

After washing three times with PT, goat anti-mouse IgE-HRP (Southern Biotech, Cat. No. 1110-05) was used as a detection reagent at 1/2000 and incubated for 1 hour at room temperature. After washing three times, ELISA reactions were revealed using TMB substrate according to the manufacturer's instructions. After 5-10 minutes at room temperature, the reaction was stopped with 1M H ₂ SO ₄ .
ELISA for cytokines
●R&D (Cat. No. DY420-05) Duoset Mouse CCL11/Eotaxin ●R&D (Cat. No. DY478-05) Duoset Mouse CCL5/RANTES
●R&D (Cat. No. DY405-05) Duoset Mouse IL-5
●R&D (Catalog number DY413-05) Duoset mouse IL-13

All ELISAs were performed according to the manufacturer's included protocols.

Histopathological lung score Lungs were excised, formalin fixed, and processed for paraffin embedding. Disease scoring was performed in a blinded manner on H&E stained sections and scores were assigned as follows.
4: Abundant infiltrate and generalized spread - loss of lung structures 3: Abundant infiltrate and generalized spread - limited damage to lung structures 2: Visible as large foci Infiltration 1: Visible as small foci Infiltration 0: normal

Results Blood Response Cellular Responses As shown in the experiments in Figure 87, no disease-driving changes in lymphocytes or myeloid cells were observed in the peripheral circulation in the two experimental schedules, and GC treatment, free (Dex) or conjugated ( (INX201J) resulted in a similar decrease in B and T cells. Specifically, Figure 87 shows the change in absolute cell numbers in peripheral blood in the two experimental schedules. OVA challenge on days 14-18 (Part 1) and days 21-25 (Part 2) (SEM, one-way ANOVA, n=10 excluding naive including n=5).

Immunoglobulin Response As shown in the experiment in Figure 88, untreated OVA-challenged animals showed a dramatic increase in IgG1 and IgE as well as OVA-specific Ig compared to naive animals. Both the Dex and INX201J groups showed similar significant reductions in IgG1, IgG1 OVA-specific production. Limited to no decrease was observed in IgE and OVA-specific IgE. In particular, Figure 88 shows changes in immunoglobulin production in peripheral blood in the two experimental schedules. OVA challenge on days 14-18 (Part 1) and days 21-25 (Part 2) (SEM, one-way ANOVA, n=10 excluding naive including n=5).

Responsive Cell Responses in Bronchoalveolar Lavage As shown in the experiment in Figure 89, OVA challenge caused the recruitment of a massive inflammatory infiltrate in the bronchoalveolar space consisting of both lymphocytes and myeloid cells. INX201J treatment resulted in a similar reduction in immune infiltration when compared to Dex in both experimental schedules except for CD8 T cells. Notably, INX201J showed the same efficacy as Dex in reducing eosinophil counts (defined as CD11b+, Ly6G-, SiglecF+, CD193+). In particular, Figure 89 shows the changes in immune infiltration in the BAL in the two experimental schedules. OVA challenge on days 14-18 (Part 1) and days 21-25 (Part 2), A) Changes in bone marrow infiltration, B) Changes in lymphocyte infiltration (SEM, one-way ANOVA, censored by control group) (n=10, n=5 for the naive group), including 3 in both the Dex and INX201J groups.

Cytokine Changes The experiment in Figure 90 shows that with the exception of CCL11, which showed a limited increase after disease induction, all other cytokines evaluated showed no changes. Neither INX201J nor Dex treatment had any effect on BAL cytokine levels. Specifically, Figure 90 shows changes in cytokine levels in BAL in the two experimental schedules. OVA challenge (SEM, one-way ANOVA, two samples censored in control group, Dex group and INX201J group) on days 14-18 (first part of the experiment) and days 21-25 (second part of the experiment) n=10, n=5 for the naive group).

Lung Disease Score Loss of bronchoalveolar morphology and large scale of inflammatory cells as shown in the experiment in Figure 91 (part 1 of the experiment, SEM, one-way ANOVA, n=10, excluding naive group n=5) Significant damage was observed in the untreated lungs, including extensive recruitment. These results show that both INX201J and Dex treatments similarly significantly reduced lung injury with limited structural damage and inflammatory infiltrate.

Conclusion The experiments in Figures 87-91 provide concrete evidence that INX201J treatment has an equivalent effect as free Dex (>10 times higher dose) on:
- reducing the recruitment of inflammatory infiltrate in the bronchoalveolar lavage (BAL) - reducing damage at the histopathological level of the lungs - reducing IgG1 and more specifically anti-OVA IgG1 in the blood circulation - inducing no changes in IgE or anti-OVA IgE observed with both therapeutic approaches in the blood circulation - no changes in cytokine production observed with both therapeutic approaches in bronchoalveolar lavage. to provoke

Importantly, similar results were observed in two parts/two different schedules of these experiments.

Example 14: Effect of an exemplary anti-VISTA antibody drug conjugate on VISTA-expressing immune cells. In these experiments, an exemplary antibody-drug conjugate (ADC) INX231J and an anti-human VISTA conjugated to a glucocorticoid (GC) payload were used. The targeting specificity of monoclonal antibodies was evaluated. To monitor/confirm GC delivery and activity, transcriptional activation of FKBP5 was measured by quantitative real-time PCR (qRT-PCR) (1). These experiments were repeated in human VISTA knock-in (hVISTA KI) mice that were knocked in with human VISTA cDNA in place of the mouse VISTA gene and express human VISTA at both RNA and protein levels with the same expression pattern as mouse VISTA. did.

In particular, the effects of non-specific ADC internalization were assessed by two different approaches. First, we added human IgG1 silent conjugated to the same payload, and second, we performed the same experiment in C57Bl/6 mice that do not express the human VISTA target (mouse VISTA only). Briefly, INX231J or INX231P, human IgG1siJ, or free dexamethasone (Dex) was delivered in vivo via intraperitoneal (i.p.) injection. After 20 hours for INX231J/hIgG1siJ/INX231P and 2 hours for Dex, blood cells and spleen cells were isolated, RNA was extracted, and FKBP5 transcript levels were assessed.

The purpose of these experiments was to verify the targeting specificity of the ADC to human VISTA expressing cells/tissues compared to free dexamethasone (Dex). To monitor/confirm GC delivery and activity, transcriptional activation of FKBP5, sensitive and early GC response genes was measured by quantitative real-time PCR (qRT-PCR). In this application, we have previously shown that Dex treatment causes a dramatic increase in FKBP5 messenger RNA in VISTA-expressing cells 2-4 hours after treatment, but that the transcriptional effects disappear by 24 hours. In contrast, the effect of ADC on FKBP5 transcription is long-lasting, with peak induction at 20 hours post-treatment, although the signal is still detectable for 3 days in monocytes and 14 days in macrophages.

In these experiments, the anti-VISTA antibody INX231 with two different payloads (J and P) or free Dex was delivered in vivo via intravenous (i.v.) or intraperitoneal (i.p.) injection, respectively. It was used. Spleen cells and blood cells were isolated, RNA was extracted, and FKBP5 transcript levels were assessed. These experiments and their results are described in detail below.

Materials and Methods Experimental Design For all three studies:
Two hours before mouse euthanasia and cell isolation, Dex was injected intraperitoneally, which corresponds to peak FKBP5 induction.

To provide sufficient time for ADC treatment and peak FKBP5 induction, ADC (INX231J or INX231P or hIgG1siJ) was injected intravenously 20 hours before mouse euthanasia and cell isolation. Of note, ADC was injected intravenously to ensure more consistent delivery of large molecules.

A control group injected with PBS only was included to define the FKBP5 transcript baseline.
Test Agents and Dosage Antibodies INX231 (lot number 72928.1.a) is a humanized anti-human VISTA antibody on a human IgG1/kappa backbone with L234A/L235A/E269R/K322A silencing mutations in the Fc region.
• INX231J (lot number JZ-0556-013-1) is INX231 conjugated via full modification of the interchain disulfide, with a drug/antibody ratio (DAR) of 8.0. The linker/payload (J) consists of a protease sensitive linker with a budesonide analog payload.
• INX231P (lot number JZ-0556-017-1) is INX231 conjugated via full modification of the interchain disulfide, with a drug/antibody ratio (DAR) of 8.0. The linker/payload (P) consists of a protease sensitive linker with a budesonide analog payload.
- Human IgG1siJ (lot number JZ-0556-025-2) is an anti-RSV mAb on a human IgG1/kappa backbone with an E269R/K322A silencing mutation in the Fc region. The drug/antibody ratio is 8.0 and is conjugated via complete modification of the interchain disulfide by the J linker/payload.

Five mice in group 1 and all animals in groups 5-7 were subjected to OVA inhalation (1% OVA in PBS) for 30 minutes for 5 consecutive days from day 21 to day 25. 2 mg/Kg Dex was injected intraperitoneally daily from day 21 to day 25. 10 mg/Kg INX201J was administered intraperitoneally on days 20, 22, and 24. Treated animals were sacrificed on day 25. All ADCs were diluted in PBS and injected intravenously in a final volume of 0.2 ml to deliver specific doses.

Dexamethasone Dexamethasone sterile injection solution from Phoenix, NDC 57319-519-05 was diluted in PBS and administered as described via intraperitoneal injection.

Mice hVISTA KI mice were bred at the Center for Comparative Medicine and Research at Dartmouth, and C57Bl/6 mice were received from Jackson Laboratories (reference number 000665).

Male or female mice were enrolled at 9-15 weeks of age.

Cell Isolation After euthanasia, cardiac blood (volumes ranging from 0.3 to 0.5 ml) and spleen were collected.

Blood preparation: 6 ml of ACK buffer was added to blood for red blood cell lysis. After 5 minutes at room temperature, cells were spun down at 1500 rpm for 5 minutes and washed once in 10 ml PBS before being pelleted and resuspended directly in RNA lysis buffer.

The spleen was mechanically dissected. After passing through a 40 μm filter, the cell pellet was resuspended in RNA lysis buffer (see below).

RNA preparation and real-time PCR
Cell pellets from blood and spleen were resuspended in 0.4 ml RNA lysis buffer from the NucleoSpin® RNA Plus kit (Macherey-Nagel, number 740984). RNA was isolated according to the manufacturer's instructions and eluted in 30 or 40 ml H2O (RNase/DNase free). RNA concentration was assessed on a Nanodrop.

Reverse transcription was performed using Taqman reverse transcription reagents (number N8080234) and according to the manufacturer's instructions.

Quantitative real-time PCR was performed using the Taqman Master Mix 2X kit (no. 4369016) and Taqman primers for mouse FKBP5 (Mm00487401_m1) and mouse HPRT (Mm446968_m1) as the housekeeping gene and QuantStudy from Applied Biosystems. Run on o3 did.

Ct data were converted to ΔCt and ΔΔCt or Log2 fold change was converted to PBS.

Experiment 1
In this experiment, we evaluated the effect of human IgG1 silent control conjugated to J payload (IgG1siJ) versus INX231J and Dex on VISTA expressing tissues (blood and spleen) in hVISTA KI male mice. IgG1siJ and INX231J were administered at 5 mg/Kg (delivering 0.1 mg/Kg payload) and FKBP5 induction was measured after 20 hours, providing sufficient time for ADC processing and robust FKBP5 induction. Dex was injected at 2 mg/Kg and FKBP5 induction was measured 2 hours later.

As shown in the experiment in Figure 92, robust FKBP5 induction was observed with INX231J and Dex treatments, whereas the conjugate Ig control caused only small, non-significant changes in FKBP5 signal. More specifically, Figure 92 shows FKBP5 transcriptional activation following INX231J injection in spleen (left) and blood (right) cells. INX231J effects and hIgG1siJ were measured 20 hours after a single intravenous injection at 5 mg/Kg (delivering a payload of 0.1 mg/Kg). Dex effects were measured 2 hours after a single intraperitoneal injection at 2 mg/Kg. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus the mean of the PBS control group. (n=4 mice/group, normal one-way ANOVA compared to PBS only group).

Experiment 2
This experiment evaluated the effects of INX231P versus Dex on blood and spleen cells in C57Bl/6 male mice that do not express human VISTA. INX231P was administered at 10 mg/Kg (delivering 0.2 mg/Kg payload) and FKBP5 induction was measured 20 hours later, providing sufficient time for ADC processing and robust FKBP5 induction. Dex was injected at 2 mg/Kg and FKBP5 induction was measured 2 hours later.

As shown in the experiments in Figure 93, although robust and significant FKBP5 induction was observed upon Dex treatment, INX231P had no effect on blood and spleen cells of wild-type mice, indicating that the effect of ADC was target-driven. It showed that there is. More specifically, Figure 93 shows FKBP5 transcriptional activation following INX231P injection in C57Bl/6 mice. INX231P effects were measured 20 hours after a single intravenous injection at 10 mg/Kg (delivering a payload of 0.2 mg/Kg). Dex effects were measured 2 hours after a single intraperitoneal injection at 2 mg/Kg. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus the mean of the PBS control group. (n=4 mice/group, normal one-way ANOVA compared to PBS only group).

Experiment 3
This experiment evaluated the effects of INX231P versus Dex on blood and spleen cells in C57Bl/6 female mice that do not express human VISTA. For ADC activity, the hVISTA KI group was added as a control. INX231P was administered at 10 mg/Kg (delivering 0.2 mg/Kg payload) and FKBP5 induction was measured 20 hours later, providing sufficient time for ADC processing and robust FKBP5 induction. Dex was injected at 2 mg/Kg and FKBP5 induction was measured 2 hours later.

As shown in the experiments in Figure 94, while robust and significant FKBP5 induction is observed with Dex treatment, INX231P has no significant effect on blood and spleen cells of wild-type mice, suggesting that the effects of ADC are target-driven. It was shown that In contrast, INX231P treatment at the same dose in hVISTA KI animals resulted in a strong and significant induction of FKBP5 transcripts indicating ADC efficacy against its target population. More specifically, Figure 84 shows FKBP5 transcriptional activation following INX231P injection in C57Bl/6 or hVISTA KI mice. INX231P effects were measured 20 hours after a single intravenous injection at 10 mg/Kg (delivering a payload of 0.2 mg/Kg). Dex effects were measured 2 hours after a single intraperitoneal injection at 2 mg/Kg. FKBP5 transcript levels were measured by real-time PCR and presented as Log2 fold change versus the mean of the PBS control group. (n=4 mice/group, normal one-way ANOVA compared to PBS only group).

Conclusions The results of Experiment 1 showed that in hVISTA KI, INX231J and Dex induced robust levels of FKBP5 in spleen and blood cells, whereas the human IgG1 silent steroid conjugate control significantly reduced FKBP5 transcript levels in both tissues. Indicates little or no impact.

The results of Experiment 2 showed that in the absence of human VISTA targets, INX231P had no effect on FKBP5 transcript levels in VISTA-expressing blood cells or spleen cells in male C57Bl/6 mice, whereas free steroids showed that they induced robust levels of FKBP5.

The results of experiment 3, a repeat of experiment 2 in female C57Bl/6 mice with the addition of a positive control of hVISTA KI mice, showed that in the absence of human VISTA targets, INX231P significantly influenced FKBP5 transcription levels in VISTA-expressing blood cells or spleen cells. indicates that it has little or no effect on In contrast, INX231P at the same dose in hVISTA KI mice or Dex induced robust levels of FKBP5 in both tissues.

Collectively, the data indicate that the presence of human VISTA target is required for efficient cellular delivery of GCs by ADCs, regardless of the GC payload.

Example 15: Effect of Exemplary Anti-VISTA Antibody Drug Conjugates on Ex Vivo Monocyte Activation (Acute (1 Day)) Assessment The experiments in this example were conducted to The efficacy and pharmacodynamic range of GATE (ADC) INX231P, an anti-human VISTA monoclonal antibody conjugated to a glucocorticoid (GC) payload, was evaluated. In a previous example, we showed that transcription of the GC target gene FKBP5 is upregulated in monocytes up to 3 days after treatment, whereas the effect of free dexamethasone (Dex) on FKBP5 is undetectable at 24 hours. Indicated.

Furthermore, we developed a model that allows evaluating the potential long-term anti-inflammatory effects of ADCs on monocytes. Briefly, ADCs were delivered in vivo via intravenous (i.v.) injection, and splenic monocytes were isolated and cultured 1-7 days later. Cells were then activated with different concentrations of lipopolysaccharide (LPS), causing a dramatic increase in cytokine production over 24 hours. Dex treatment 2 hours before monocyte isolation robustly reduces cytokine production.

These experiments (Experiments 1, 2, and 3, discussed below) were performed using human VISTA with human VISTA cDNA knocked in instead of the mouse VISTA gene and with the same expression pattern as mouse VISTA at both the RNA and protein levels. was performed in human VISTA knock-in (hVISTA KI) mice expressing hVISTA KI. The purpose of these studies was to evaluate the effects of INX231P in vivo treatment on monocytes expressing high levels of VISTA. The second objective was to compare its anti-inflammatory potential with the agonist counterpart INX901. Briefly, ADCs were delivered in vivo via intravenous (i.v.) injection, and splenic monocytes were isolated and cultured 1-7 days later. Cells were then activated with different concentrations of LPS and supernatants were collected at 24 hours to assess cytokine responses (Luminex Mouse 32-plex (Experiment 1) or ELISA (Experiment 2 and Experiment 1) for selected cytokines. 3)).

Materials and Methods For all three experiments, Dex was injected intraperitoneally 2 hours before mouse euthanasia and cell isolation with optimal response. In experiments 2 and 3, both ADC INX231P and the agonist counterpart INX901 were injected intravenously 24 hours before mouse euthanasia and cell isolation to provide sufficient time for ADC treatment. A control group injected with PBS was also included to define the maximal cytokine response.
Test Agents and Dosage Antibodies INX231 (lot number 72928.1.a) is a humanized anti-human VISTA antibody on a human IgG1/kappa backbone with L234A/L235A/E269R/K322A silencing mutations in the Fc region.
• INX231P (lot number JZ-0556-017-1) is INX231 conjugated via full modification of the interchain disulfide, with a drug/antibody ratio (DAR) of 8.0. The linker/payload (P) consists of a protease sensitive linker with a budesonide analog payload.
-INX901 (lot number BP-021-016-23) is a humanized anti-human VISTA antibody on a human IgG2/kappa backbone.

All antibodies and ADCs were diluted in PBS and injected intravenously (i.v.) in a volume of 0.2 ml to deliver specific doses.

Dexamethasone Dexamethasone sterile injection solution from Phoenix, NDC 57319-519-05 was diluted in PBS in a volume of 0.2 ml and administered as described via intraperitoneal (i.p.) injection.

Mice hVISTA KI mice were bred at the Center for Comparative Medicine and Research at Dartmouth, and C57Bl/6 mice were received from Jackson Laboratories (reference number 000665). Male or female mice were enrolled at 9-15 weeks of age.

For splenic monocyte isolation experiments 1 and 2, cells were isolated using the EasySep™ Mouse Monocyte Isolation Kit from StemCell (Cat. No. 19861) and ADC-INVIVO-109 according to the manufacturer's instructions. used the monocyte isolation kit from Miltenyi (catalog number 130-100-629). Similar cell numbers and purity were obtained across experiments.

Ex Vivo LPS Stimulation Assay After counting, cells were plated at approximately 100,000 cells/well, depending on the number of isolated cells and as singles (all data reported were plated (note normalized to cell number). LPS was added to tissue culture media at 0, 10, or 100 ng/ml as described. Cell supernatants were collected at 24 hours for cytokine analysis.

Cytokine analysis experiment 1:
Cytokine analysis was performed on 25 μl of supernatant using a Millipore mouse 32-plex platform and a Shared Resou at the Immune Monitoring Lab (IML, Dartmouth-Hitchcock Norris Cotton Cancer Center). rces) performed the analysis. For a description of all protocols and analyses, please refer to the following website: http://www. dartmouth. edu/～dartlab/? page=multiplexed-cytokines.

Experiments 2 and 3:
Cytokine analysis was performed via ELISA for TNFα, MIP-1a, and MIP-1b using the following kits.
○Mouse CCL3/MIP-1 Alpha DuoSet ELISA (R&D, No. DY450-05)
○Mouse CCL3/MIP-1 Beta DuoSet ELISA (R&D, No. DY451-05)
○Mouse TNFα ELISA (Biolegend, catalog number 430904)

All ELISAs were performed according to the manufacturer's instructions.

Results Experiment 1: Effect of Dexamethasone on Ex Vivo LPS Stimulation of Monocytes Isolated from Spleen In Experiment 1, the effect of in vivo treatment with two different doses of Dex on ex vivo splenic monocytes was evaluated. Briefly, female C57Bl/6 mice were treated with 2 or 0.2 mg/Kg Dex by intraperitoneal injection. The control group received PBS. Two hours later, the animals were sacrificed and the spleens were collected. Monocytes were isolated and cultured. Due to low monocyte counts after isolation, 5 samples per group were pooled into 2 samples for plating (pool of 2 or 3 initial samples). Subsequent cytokine data were then normalized to cell number.

After plating, cells were treated with 10 or 100 ng/ml LPS or left untreated. Cell supernatants were collected at 30 minutes and 24 hours. Cytokine production was analyzed on mouse 32-plexes. No changes in cytokine levels were observed at 30 minutes (not shown). At 24 hours, eight cytokines G-CSF, IL-6, IL-10, IP-10, MIP-1a, MIP-1b, TNFα, and RANTES were upregulated by LPS treatment in spleen samples. As shown in Figure 95, Dex in vivo treatment resulted in reduced cytokine responses at both LPS concentrations. Figure 95 shows that in vivo Dex treatment induces a substantial reduction in ex vivo monocyte inflammatory responses to LPS. In experiments, mice were injected intraperitoneally with PBS or Dex at 2 mg/Kg or 0.2 mg/Kg. Two hours later, splenic monocytes were isolated, cultured, and stimulated with LPS at 0, 10, and 100 ng/ml. 24-hour supernatants were analyzed on a Luminex 32-plex (n=5 mice/group, but samples 1, 2, 3, and 4, 5 were pooled into two samples).

Experiment 2: Effect of dexamethasone vs. INX231P vs. INX901 on ex vivo LPS stimulation of monocytes isolated from spleen. , the effects on splenic monocytes from hVISTA KI female mice stimulated ex vivo with LPS were evaluated. To evaluate the pharmacodynamic range of these molecules, we tested 24 hours, 3 days, and 7 days post-treatment for both INX231P and INX901-treated groups, and 2 hours, 2 days, and 6 days post-treatment for the Dex-treated group. At one day, splenic monocytes were isolated. INX231P and INX901 were administered at 10 mg/Kg and Dex was injected at 2 mg/Kg. After plating, samples were treated with 10 ng/ml LPS or left untreated. Cell supernatants were collected at 24 hours. Cytokine analysis was performed for NFα, MIP-1b, and MIP-1a via ELISA. Cytokine data were normalized to the number of cells plated.

As shown in the experiment in Figure 96, INX231P at day 1 had a robust effect on TNFα and MIP-1b production, comparable to Dex at 2 hours. No effect was observed at later time points. In contrast, INX901 had no effect (MIP-1a and b) or increased (TNFα) the cytokines analyzed. More specifically, Figure 96 shows the effect of in vivo treatment with INX231P on ex vivo monocyte inflammatory responses to LPS. Mice were injected intraperitoneally with PBS or Dex at 2 mg/Kg 2 hours, 2 days, or 6 days before cell isolation and at 10 mg/Kg 1 day, 3 days, and 7 days before cell isolation. INX231P and INX901 were injected intravenously. After isolation, splenic monocytes were cultured and subjected to LPS stimulation at 0 or 10 ng/ml (only 10 ng/ml is shown). 24-hour supernatants were analyzed by ELISA (n=4 mice/group, one-way ANOVA comparing to PBS-treated group was performed only on day 1 (D1) samples).

Experiment 3: Effect of dexamethasone vs. INX231P vs. INX901 on ex vivo LPS stimulation of monocytes isolated from spleen. Cytokine responses were assessed only after hours. Splenic monocytes were isolated, cultured, and treated with 10 or 100 ng/ml LPS. Cell supernatants were collected at 24 hours. Cytokine analysis was performed for TNFα, MIP-1b, and MIP-1a via ELISA, and data were normalized to the number of cells plated.

The experiments in Figure 97 show that INX231P potently prevented monocyte ex vivo activation of all three cytokines analyzed at both LPS concentrations. Note that when cells were stimulated with 100 ng/ml LPS, Dex treatment appeared to lose efficacy, suggesting that INX231P is more potent while delivering 10 times less payload. Finally, as observed in Experiment 3, INX901 treatment had no effect on LPS-induced cytokine responses. More specifically, Figure 97 shows in vivo treatment with INX231P on ex vivo monocyte inflammatory responses to LPS. Mice were injected intraperitoneally with PBS or Dex at 2 mg/Kg 2 hours before cell isolation and intravenously with INX231P and INX901 at 10 mg/Kg 24 hours before cell isolation. Splenic monocytes were cultured and stimulated with LPS at 10 and 100 ng/ml. 24 hour supernatants were analyzed by ELISA (n=4 mice/group, separate routine one-way ANOVA compared to PBS treated group for each LPS dose).

Conclusion Experiment 1 showed that 2 mg/Kg in vivo Dex treatment efficiently prevented ex vivo monocyte activation by LPS as shown by a dramatic reduction in cytokine production. Experiment 2 showed that INX231P treatment in vivo was able to reduce ex vivo activation of monocytes, as shown by a reduction in some cytokine production at 24 hours, but these effects were This is consistent with the known half-life of monocytes, which is in the range of 2-3 days. In addition, the effect of ADC on cytokine production is due to GC delivery to VISTA-expressing cells, as treatment with unconjugated agonist-enabled antibodies (same CDR) has no anti-inflammatory activity. Experiment 3, a repeat of experiment 2, except looking only 2 hours after Dex treatment or 24 hours after ADC and unconjugated agonist treatment, showed that while INX231P strongly reduced ex vivo activation of monocytes, , indicating that the agonist antibody had no effect.

Therefore, the experimental results show the following.
• LPS-induced cytokine responses on isolated splenic monocytes are efficiently controlled by dexamethasone.
• INX231P, but not INX901, therapy efficiently controls LPS-induced cytokine responses on isolated splenic monocytes when administered as early as 24 hours in vivo. By day 3, no effects are observed consistent with the known half-life of murine monocytes, which ranges from 2 to 3 days.
• INX231P, but not INX901, potently prevents LPS-induced ex vivo activation of splenic monocytes. In this experiment, INX231P delivers 10 times less GC payload than free Dex, but shows high potency at high stimulation levels (100 ng/ml LPS), whereas Dex appears to lose efficacy. I took note. Finally, as observed in Experiment 3, INX901 treatment had no effect on LPS-induced cytokine responses.

Overall, the experimental results show that INX231P in vivo treatment is able to prevent monocyte ex vivo activation with at least 10 times greater potency than free steroids. In contrast, the agonist anti-VISTA antibody INX901 showed no efficacy in this model. Therefore, the results observed in this experiment are induced entirely by the steroid payload and not by VISTA modulation.

Example 16: Effects of anti-VISTA drug conjugates on transcription in monocytes, T regs, and B cells are dependent on target expression.
Here we describe anti-human VISTA monoclonal antibodies conjugated to various glucocorticoid (GC) payloads and their in vitro and in vivo effects. This example evaluates 1) the effect of INX201J on monocytes and B cells versus isotype control (huIgG1si J) and free J payload, and 2) the effect on INX231P (on Tregs) versus free payload (INX-SM-3). VISTA target dependence is assessed by evaluating the effect on transcription of the GC reporter gene FKBP5 for exemplary ADCs according to the invention.

As shown herein, treatment with anti-VISTA steroid ADCs resulted in a robust and dose-dependent upregulation of FKBP5 on monocytes, cells with high expression levels of VISTA. A significant but more moderate effect was observed in Tregs, which have lower VISTA expression than monocytes. A negligible effect was seen on B cells in which VISTA was not expressed. No changes in FKBP5 expression were observed in either monocytes or B cells when treated with the steroid conjugate isotype control.

Antibody drug conjugates (ADCs) enable specific cellular targeting of highly potent drugs to enable efficacy while limiting toxicity. INX201 and INX231 are anti-human VISTA antibodies. In their steroid-conjugated form, INX201J and INX231P are expected to deliver steroids to VISTA-expressing cells, including myeloid cells, and T cells, with little or no effect on VISTA-negative cells such as B cells. (Cancer Res. 74:1924-1932, 2014).

To monitor/confirm GC delivery and activity, transcriptional activation of FKBP5, a direct and robust biomarker of glucocorticoid activity, was measured by quantitative real-time PCR (qRT-PCR) (JCEM 101:4305- 4312, 2016). This evaluation was performed on isolated human monocytes, regulatory T cells (T regs), and B cells after in vitro treatment with ADCs and ADCs.

Materials and Methods Monocytes or B cells were isolated from healthy donor blood samples and treated with free steroids, anti-VISTA conjugated steroids, or conjugated isotype controls. RNA was isolated and changes in FKBP5 transcript levels were assessed by qPCR.

For monocyte versus B cell analysis, one blood donor collection was used for single drug concentration experiments, and blood from another single donor collection was used to assess drug dose response. Blood from two separate donors was used for regulatory T cell (Treg) analysis.
Test Agent ●Free J payload, INX J-2 (Abzena). INX J-2, or simply Free J payload, is a patent-reported budesonide analog utilized in the complete linker/payload INX J.
●INX201 (Aragen, lot number BP-3200-019-6) is a humanized anti-human VISTA on a human IgG1/kappa backbone with V234A/G237A/P238S/H268A/V309L/A330S/P331S silencing mutations in the Fc region. It is an antibody.
INX201J (Abzena, lot number JZ-0556-025-1) is an INX201 antibody that is conjugated via full interchain disulfide modification and has a drug/antibody ratio (DAR) of 8.0. . Linker/payload (J) is based on previously reported linker/payload. It consists of a protease sensitive linker with a budesonide analogue payload.
• INX-SM-3 (O2H) is a budesonide analog payload utilized in the linker/payload INX P.
- INX231 (ATUM, lot number 72928.1.a) is a humanized anti-human VISTA antibody on a human IgG1/kappa backbone with L234A/L235A/E269R/K322A silencing mutations in the Fc region.
INX231P (Abzena, lot number JZ-0556-017-1) is a linker/payload consisting of a protease-sensitive linker with INX-SM-3 through complete modification of interchain disulfides with a DAR of 8.0. INX231 conjugated to INX P.
• Human IgG1siJ (Abzena, lot number JZ-0556-025-2) is an isotype control on the human IgG1/kappa scaffold with an E269R/K322A silencing mutation in the Fc region. The DAR is 8.0 and is conjugated via full modification of the interchain disulfide with the INX J linker/payload.
Additional reagents Ficoll-Paque Plus (GE Healthcare, catalog number 17-1440-03)
●RPMI 1640 (VWR, catalog number 16750-084) without L-glutamine
●Penicillin/Streptomycin/Glutamine (ThermoFisher, catalog number 10378016)
●1M Hepes (Gibco, catalog number 15630-080)
●Human AB serum (Valley Biomedical, catalog number HP1022HI)

Preparation of PBMCs Human PBMCs were isolated under sterile conditions from apheresis cones obtained from the blood donor program at Dartmouth Hitchcock Medical Center from de-identified healthy human donors.

Blood was transferred to a 50 ml Falcon tube and diluted to 30 ml with PBS. 13 ml of Histopaque 1077 (Sigma Aldrich) was slowly layered under the blood and the tube was centrifuged at 850 x g for 20 minutes at room temperature without interruption at gentle acceleration.

Mononuclear cells were collected from the plasma/Ficoll interface, resuspended in 50 ml of PBS, and centrifuged at 300 xg for 5 minutes. Cells were resuspended in PBS and counted.

Assay Protocol Various immune populations were isolated using different cell isolation kits according to the manufacturer's instructions.
●EasySep human monocyte enrichment kit (no CD16 depletion) (StemCell, catalog number 19058)
●Pan B cell isolation kit, human (Miltenyi Biotec, 130-101-638)
●EasySep™ Human CD4+ CD127 Low CD49d- Regulatory T Cell Enrichment Kit (StemCell, Catalog No. 19232)

Monocytes, B cells, or Tregs (from a single donor) were cultured at 2 ＾6 cells were plated.

In single-dose experiments, cells were treated with 20 nM of free J payload or INX-SM-3 payload or molar payload equivalent to huIgG1si J, INX201J, or INX231P (bound form of INX-SM-3).

For dose response, serial dilutions resulting in free J payload of 100, 20, 5, 0.5, 0 nM or molar payload equivalent to INX201J. For the 0 nM point, unconjugated INX201 was used equivalent to the amount of antibody used for a 100 nM molar payload equivalent to INX201J (eg, 12.5 nM unconjugated antibody). Wells without treatment were used as controls.

Plates were incubated at 37°C for 1 day.

Cells were then harvested and wells for each condition were pooled after harvest to allow sufficient RNA for subsequent qRT-PCR analysis.

RNA preparation and real-time PCR
After washing once with PBS, RNA was isolated from the cell pellet using either the RNeasy Plus Mini kit (Qiagen, PN: 74136) or the NucleoSpin RNA Plus (Macherey-Nagel, no. 740984.250). RNA was isolated according to the manufacturer's instructions and eluted in 30 or 40 μl H2O (RNase/DNase free). RNA concentration was assessed by UV spectroscopy using a Nanodrop 2000.

Reverse transcription was performed using Taqman reverse transcription reagents (number N8080234) and according to the manufacturer's instructions.

Quantitative real-time PCR was performed using the Taqman Master Mix 2X kit (number 4369016) and run on a QuantStudio3 from Applied Biosystem. Primer used:
●Experiment 1 and Experiment 2
i. Life Technologies, catalog number 433111182 Hs01561006_m1 (FKBP5)
ii. Life Technologies, catalog number HS99999905_m1 (GapDH)
●Experiment 3 and Experiment 4
i. TaqMan gene expression assay (FAM-MGB); Assay ID: Hs01561006_m1 (FKBP5)
ii. TaqMan gene expression assay (FAM-MGB); Assay ID: Hs01922876_u1 (GapDH)

Ct data were converted to ΔCt and ΔΔCt or Log2 fold change was compared to untreated controls.

Results Experiment 1
This experiment evaluated the need for targeted expression for steroid delivery by ADCs, as assessed by the induction of FKBP5 transcription in monocytes as the VISTA-positive cell population and B cells as the VISTA-negative population. Free steroids were added as a positive control for the effect of steroids on FKBP5 levels in specific cell types. Free steroid (free J payload), J linker payload conjugate anti-VISTA (INX201J) or isotype control (huIgG1si J) were administered to provide the same molar equivalent of payload (20 nM).

As shown in Figure 98, a robust increase in FKBP5 transcription was observed with free J payload in both monocytes and B cells compared to untreated controls. However, when treated with anti-VISTA conjugate payload (INX201J), robust FKBP5 transcription was observed in monocytes but not in B cells. No FKBP5 transcription was detected in both cell types when treated with payload conjugate isotype control (HuIgG1si). In particular, Figure 98 shows FKBP5 transcriptional activation in B cells or monocytes in cells with 20 nM free J payload, or payload conjugated to equimolar amounts of INX201 (INX201J) or isotype control (huIgG1siJ). It was treated with. Transcript levels were analyzed as technical duplicates.

Experiment 2
The experiment in Figure 99 extended Experiment 1 by evaluating the dose-dependent effect of treatment with steroid-conjugated anti-VISTA ((INX201J) on monocytes (high VISTA expression). ). At the 0 nM concentration alone, the INX201 unconjugated antibody was treated with the same amount of antibody present for the 100 nM payload sample. Specifically, the 12.5 nM ADC was treated with the same amount of antibody as was present for the 100 nM payload sample. To deliver the payload, unconjugated INX201 was added to 12.5 nM for the 0 nM sample. As shown in Figure 99, treatment of monocytes with INX201J results in a robust dose-dependent effect. , FKBP5 transcriptional activation in monocytes was demonstrated in cells treated with increasing amounts of INX201J [0-100 nM payload]). 0 payload represents treatment with unconjugated INX201 antibody alone at the same amount of antibody as the 100 nM payload INX201J dose. Transcript levels were analyzed as technical duplicates.

Experiment 3
In the experiment in Figure 100, the effect of a second anti-VISTA steroid conjugate (INX231P) on FKBP5 transcriptional induction in VISTA-expressing Tregs was evaluated. As shown in Figure 100, treatment of Tregs with a molar payload equivalent to 20 nM free payload (INX-SM-3) or anti-VISTA conjugated payload (INX231P) resulted in an increase in FKBP5 transcription. This experiment was performed with two different donors and the isolated Treg purity was ≧75%.

Experiment 4
In the experiment in Figure 101, the effect of anti-VISTA steroid conjugate (INX201J) conjugated with the same linker/payload (huIgG1si J) versus isotype control on FKBP5 transcriptional induction in Tregs was evaluated. As shown with INX201J treatment, increasing −1/delta Ct represents an increase in transcript abundance compared to housekeeping (GapDH). Treatment of Tregs with INX201J delivering a 20 nM steroid payload results in a 2.1-fold increase in FKBP5 transcription vs. conjugate isotype control (fold change = 2^(ΔCt INX201J - ΔCt huIgG1si J)). This experiment was performed with one donor and the isolated Treg purity was ≧75%. Specifically, the data in Figure 101 shows FKBP5 induction in T regs from one donor treated with 20 nM payload equivalents of INX201J compared to 20 nM payload equivalents of huIgG1si J. Samples were analyzed as technical duplicates. The purity of isolated Tregs was ≧75% as assessed by flow cytometry.

Conclusions Data show that anti-VISTA antibodies conjugated to steroids specifically induce FKBP5 transcription in monocytes and Tregs, but not in B cells, indicating that payload delivery is specific and target-dependent. Show that.

All cell types analyzed showed robust responses to free payload, but only VISTA-expressing cell types (monocytes/Tregs) showed a moderate to strong response when treated with 20 nM anti-VISTA steroid conjugate. Show response. In addition, isotype control ADCs showed little or no induction of FKBP5 when compared to no treatment controls.

The targeting requirement for GC effects is supported by robust dose-dependent effects on VISTA-expressing cells and limited or no effects on non-VISTA-expressing cells by anti-VISTA ADCs.

Example 17: RNA Expression of Various Immune Cells by Antigens Targeted by Exemplary Anti-Inflammatory Drug Conjugates As described above, the subject anti-inflammatory drug conjugates are targeted by previous anti-inflammatory drug conjugates. Due to the expression or lack of expression of VISTA on specific immune and non-immune cells compared to the antigens identified, it is believed to have superior attributes with respect to previous anti-inflammatory drug conjugates.

This is suggested by the reported RNA expression profiles. In particular, the inventors first described the "Human Protein Atlas Version 20.1" and Berglund L et al. , “A genetic Human Protein Atlas for expression profiles based on antibodies”, Mol Cell Proteomics, Vol. 7(10):2019-2027 (October 1, 2008) (https://www.proteinatlas.org) RNA expression of VISTA and other immune cell targets on immune and non-immune cells compared.

Based on this analysis, we prepared a consensus dataset from Human Protein Atlas version 20.1 and human tissue/cell RNAseq data reported from Berglund et al (Id). The results of this comparison are shown in FIG. In particular, FIG. 102 shows that TPM<10 represents (minimal/no expression "-"), TPM 10-100 represents (low/medium expression "+"), and TPM>100 represents (high expression "+"). ++”) summarizes the consensus RNA expression levels by different cells for VISTA and other ADC targets (CD40, TNF, PRLR, CD174) based on reported “transcripts per million” (TPM). . As known in the art, TPM is a well-known normalization method for RNA-seq, which states that "for every 1,000,000 RNA molecules in an RNA-seq sample, x is "derived from a gene/transcript."

As shown in Figure 102, VISTA is widely expressed on activated and non-activated myeloid cells (monocytes, macrophages, neutrophils), T cells, dendritic cells, NK cells, and eosinophils. (not shown in the data, e.g. “The immune checkpoint molecule VISTA regulates allergen-specific Th2-mediated immune responses”, Tatsukuni O. hno et al., International Immunology, Volume 30, Issue 1, (see January 2018, Pages 3-11), in contrast, TNF expression is relatively low on most cell types and is even expressed only on activated immune cells, and CD163 is expressed by myeloid cells but not in lymphoid cells. CD40 is not expressed by cells, CD40 is deficient in T cells, PRLR is not widely expressed on immune cells and is not immunorestricted, and CD74 is deficient in neutrophils. (This is because neutrophils are important in the early (acute) stages of inflammation, especially during bacterial infections, environmental exposures, and some cancers, and are actually directed toward sites of inflammation via chemotaxis. (Yoo SK et al., (November 2011). “Lyn is a redox sensor that mediates leukocyte wound attraction in v. ivo”, Nature, 480(7375):109-12).

Regarding the above, although these reported RNA expression levels by different immune cells are of interest, they do not provide actual evidence regarding the relative putative efficacy of these antigens as ADC targets. Rather, this depends on the actual surface protein expression levels of these targets on different immune cells and the experimental evidence that VISTA ADCs effectively target and are effective within different immune cells (i.e., these different internalization and release of a therapeutically effective amount of an active inflammatory agent, such as a steroid, into one or more of the types of immune cells).

Example 18: Comparison of Anti-VISTA, Anti-CD74, Anti-CD163, and Anti-mTNFα Antibodies to Antigen Targeted by Exemplary Anti-inflammatory Drug Conjugates and Antibody Binding Capacity on Human Peripheral Blood Mononuclear Cells and Whole Blood Comparison of surface expression of VISTA by various immune cells. Surface antigen density of VISTA, CD74, CD163, and membrane TNFα (mTNFα) was determined by flow cytometry on naive human peripheral blood mononuclear cells (PBMCs) and in whole blood. Evaluated by. As shown below, the data show the following when compared to CD74, CD163, and mTNFα.
• Only VISTA is expressed at steady state on human CD8+ and CD4+ T cells • VISTA shows the highest antigen density on CD14+ monocytes • Surface mTNFα was not detected on any cell type tested.

VISTA is highly expressed on most hematopoietic cells, especially myeloid cells and T cells. The purpose of this study was to evaluate the antigen density of VISTA, CD74, CD163, and mTNFα in both human PBMC and leukocytes derived from whole blood.

Materials and Methods Experimental Design Binding of directly labeled antibodies to human cells (PBMC) or whole blood leukocytes from multiple donors was determined by flow cytometry and antigen density was calculated using calibration beads.

Reagent antibody:
Anti-VISTA GG8 (Aragen, lot number AB131122-3) is a chimeric anti-human VISTA antibody on a wild-type human IgG1/kappa backbone and was produced at ImmuNext. The GG8 clone was conjugated with Alexa Fluor 647 dye (Invitrogen, catalog number A20186) according to the manufacturer's instructions for labeling and purification. Unless otherwise specified, all remaining antibodies were purchased from BioLegend and used as received, including:
CD127 Brilliant Violet 421 clone A019D5,
CD14 PE-Cy7 clone M5E2,
CD20 Brilliant Violet 510 clone 2H7,
CD4 APC-Cy7 clone OKT4,
CD163 Alexa Fluor 647 clone GHI/61,
CD25 FITC clone BC96,
CD74 Alexa Fluor 647 clone 332516 (R&D Systems),
CD8 PE clone BW135/80 (Miltenyi), mTNFα Alexa Fluor 647 clone mAb11.

Other reagents:
Calibration beads (Quantum Simply Cellular Mouse IgG) were purchased from Bangs Laboratories and used according to the manufacturer's protocol.

PBMC Preparation Human PBMC were isolated under sterile conditions from apheresis cones obtained from the blood donor program at Dartmouth Hitchcock Medical Center from healthy unrelated human donors. First, blood was transferred to a 50 ml Falcon tube and diluted to 30 ml with PBS. 13 ml of Histopaque 1077 (Sigma Aldrich) was slowly layered under the blood and the tube was centrifuged at 850 x g for 20 minutes at room temperature without interruption at gentle acceleration.

Mononuclear cells were collected from the plasma/Ficoll interface, resuspended in 50 ml of PBS, and centrifuged at 300 xg for 5 minutes. Cells were resuspended in PBS and counted.

Whole Blood Preparation Fresh blood was collected from healthy unrelated human donors at the Dartmouth Hitchcock Medical Center and staining was performed on whole blood.

Antibody Binding and Analysis PBMC Staining PBMC were resuspended in PBS/0.2% BSA buffer containing human Fc blocking reagent (eBioscience, 14-9161-73) and then 106 cells/well were plated in 96-well plates. distributed. An antibody cocktail was prepared and PBMCs were stained on ice for 30 minutes to limit internalization and washed twice with PBS.

Whole blood staining 100 μl of blood was stained in a deep-well 96-well plate and the antibody cocktail was added directly. After 30 minutes of incubation, red blood cells were lysed with 1 ml of ACK buffer (Gibco) for 10 minutes. Blood was centrifuged and blood leukocytes were transferred to 96-well plates, washed with PBS, and analyzed.

Binding Quantification Quantification beads were stained with anti-VISTA, anti-CD74, anti-CD163, and anti-mTNFα according to the manufacturer's protocol. Cells and beads were analyzed by fluorescence associated cell sorting (FACS) using a Macsquant (Miltenyi) flow cytometer and FlowJo for analysis. Antibody binding capacity was calculated using the QuickCal analysis template provided with the Quantum beads.

All graphs were created with GraphPad (Prism).

Results Evaluation of test antibody binding on PBMCs To evaluate antigen density on cell populations, human PBMCs from five different donors were incubated with mAbs and analyzed by flow cytometry. Median fluorescence was normalized by excluding background signal and calibrated against quantification beads with known antibody binding capacity. Cell populations were identified as CD20 ⁺ B cells, CD14 ⁺ SSC ^high monocytes, CD8 ⁺ and CD4 ⁺ T cells, and CD4 ⁺ CD25 ⁺ CD127 ^low T regulatory cells (T reg). All values are reported as mean±SD.

As shown in Figure 103A, CD14+ monocytes express three targets at high levels, with VISTA being the most abundant with antibody binding capacity or ABC=111587±30502, followed by CD74 (ABC= 52001±4765), and CD163 (ABC=36671±12339) (FIG. 103A). Of note, for CD163, one outlier donor showed 5-fold higher expression than the remaining four, resulting in an elevated mean value.

As shown in Figure 103B, only CD74 was detected on B cells, and 69574±14997 molecules were quantified. VISTA was the only protein expressed on non-activated T cells, with 5938 ± 3113 molecules on CD4 ⁺ (Figure 103C), 6641 ± 4059 on T reg (Figure 103D), and 9958 molecules on CD8 ⁺ It can be seen that the average density was ±2741 molecules (Figure 103E).

In naïve PBMCs, mTNFα was not detected above background levels. The absence of mTNFa was confirmed by negative staining with a second mTNFα antibody (R&D Systems, biosimilar of adalimumab, clone Hu7). mTNFa was also not detected in LPS-activated cells (data not shown). The specificity of commercially available anti-TNFa antibodies was determined by the manufacturer and confirmed internally via ELISA (data not shown).

Figures 103A-E summarize the quantification of antigen density of VISTA, CD74, CD163, and mTNFα on identified cell populations; A) monocytes express VISTA, CD74, and CD163; B) B) Cells express CD74, C) CD4+ T cells, D) CD4+ T reg, E) CD8+ T cells express VISTA (mean±SD, n=5 donors).

Analysis of antibody binding to whole blood leukocytes Neutrophils are an integral part of the immune system missing from PBMC preparations. Therefore, whole blood leukocytes from three healthy donors were also investigated to assess antigen expression on the cell populations. Similar to PBMC, whole blood was stained with a monoclonal antibody cocktail and analyzed by FACS. Median fluorescence was normalized by excluding background signal and calibrated against quantification beads with known antibody binding capacity.

Cell populations were identified as CD20 ⁺ B cells, CD14 ⁺ SSC ^high monocytes, CD66b ⁺ SSC ^high neutrophils, CD8 ⁺ and CD4 ⁺ T cells, and CD4 ⁺ CD25 ⁺ CD127 ^low T regulatory cells (T reg). . All values are reported as mean±SD.

As observed in PBMCs, VISTA was most abundant on CD14 ⁺ monocytes (ABC=223674±16503) and CD163 expression was maintained at 13126±790 molecules, whereas CD74 expression was It was much lower than PBMC (ABC=562±338) (Figure 104A). Expression of CD74 on CD20 ⁺ B cells was highly variable between donors (5800±3121). A similarly minimal signal was observed for VISTA (ABC=1280±291) (Figure 104B). Neutrophils showed high levels of VISTA expression (ABC=68571±14731) (Figure 104C), but no other targets of interest were detected. Finally, VISTA expression on T cells was also confirmed in whole blood, with 8717±886 molecules detected. VISTA expression on T cells was also confirmed in whole blood, with 8717±886 molecules on CD4 ⁺ (Figure 104D), 7486±1767 on T reg (Figure 104E), and 5012±2438 on CD8+ (Figure 104F). was detected. Figures 104A-F summarize the quantification of antigen density of VISTA, CD74, CD163, and mTNFα on identified cell populations in human blood; A) Monocytes express VISTA, CD74, and CD163. , B) B cells express CD74, C) Neutrophils express VISTA, D) CD4+ T cells, E) CD4+ T reg, F) CD8+ T cells express VISTA (mean ± SD, n =3).

VISTA expression on activated immune cells (monocytes) compared to other targets Additionally, experiments were performed comparing the expression of VISTA to other antigens on activated immune cells (monocytes). As shown in Figure 110, expression of VISTA on activated immune cells (monocytes) can be suppressed by targeting with other proteins (especially other steroid ADCs) on activated immune cells (monocytes). The expression level of protein) was compared.

In experiments, human whole blood from healthy donors was activated with LPS (100 μL per well in a U-bottom 96-well plate, 1 μg/mL LPS, 2 hours at 37° C.). Cell surface protein expression levels on activated immune cells were assessed by flow cytometry. Directly conjugated antibodies were used for restaining and included anti-VISTA clone GG8, CD163 clone GHI/61, CD74 clone 332516, and mTNFα clone mAb11. [Anti-VISTA clone GG8 (Aragen, Lot No. AB131122-3) is a chimeric anti-human VISTA antibody on a wild-type human IgG1/kappa backbone, produced at ImmuNext, and the GG8 clone manufactured for labeling and purification. It was conjugated with Alexa Fluor 647 dye according to the manufacturer's instructions (Invitrogen, catalog number A20186). All remaining antibodies were purchased and used as received: CD163 Alexa Fluor 647 clone GHI/61 (Biolegend), CD74 Alexa Fluor 647 clone 332516 (R&D Systems), mTNFα Alexa Fluor 647 clone mAb11 (B iolegend)].

As shown, in Figure 103, the VISTA expression pattern on activated monocytes was similar to the expression levels observed on non-activated monocytes, while other detections detected on activated monocytes The expression levels of these proteins were low. Furthermore, mTNFα MFI was only slightly higher than fluorescence minus one (FMO) control. These results demonstrate that the subject VISTA ADCs are effective against both activated and non-activated immune cells, such as monocytes and other myeloid cells that express VISTA at high levels (as well as those previously identified). Targeting other immune cells that constitutively express VISTA (e.g. eosinophils, dendritic cells, macrophages, myeloid cells, CD4 T cells, CD8 T cells, Tregs, NK cells, monocytes, neutrophils) It is further shown that it can be used to

Conclusion The data summarized in Figures 103, 104, 110 and Table 6 below demonstrate the following.
●Human VISTA is the most robust ADC target protein with high expression levels on monocytes, neutrophils, and T cells. Of note, although several RNA databases have described high levels of CD74 transcripts in T cells (Berglund L et al., “A genetic Human Protein Atlas for expression profiles based on antibod ies”, Mol Cell Proteomics. (2008) DOI: 10.1074/mcp.R800013-MCP200), no surface expression of CD74 on T cells was observed.
VISTA is expressed at much higher levels on activated immune cells (monocytes) than other targets analyzed, and VISTA on activated and non-activated immune cells (e.g. monocytes) Expression is similar and the subject VISTA ADCs are effective for both activated and non-activated immune cells, such as monocytes (as well as eosinophils, NK cells, macrophages, CD4 T cells, CD8 T cells). We further show that it can be used to target other previously identified immune cells such as , Tregs, NK cells, neutrophils).
• CD74 is consistently detected on both PBMC and whole blood B cells, but only on monocytes from PBMC.
●CD163 is expressed only on monocytes from PBMC.
• mTNFα was not detected in any cell population analyzed.
VISTA is the only protein expressed on non-activated (naïve) T cells, with 5938±3113 molecules on CD4 ⁺ cells, 6641±4059 on T reg, and 9958±2741 molecules on CD8 ⁺ cells. is the average density of molecules.

Table 6 contains a summary of the surface expression of different antigens, including VISTA, on human immune cell populations. Expression of analyzed surface targets was classified as present (light gray) or absent (dark gray) on the cell surface. Based on normalization to quantification beads, + corresponds to 1000-10000 molecules, ++ corresponds to 10000-100000, +++ corresponds to >100000, WB - whole blood, PBMCS - peripheral blood. Mononuclear cells, na - not applicable.

As mentioned above, the subject anti-inflammatory drug conjugates reduce the expression or lack of expression of VISTA on certain immune and non-immune cells compared to antigens targeted by previous anti-inflammatory drug conjugates. Therefore, it is believed to have superior attributes with respect to previous anti-inflammatory drug conjugates.

Based on these results, VISTA is expressed only by immune cells and therefore, unlike some other targets such as PRLR, which is not immunorestricted, VISTA ADC induces toxicity towards non-target cells. The trend should be low. Furthermore, VISTA is constitutively expressed by naïve immune cells and especially T cells, so unlike some other ADC targets such as TNF, VISTA ADC has a certain level of efficacy (i.e., activation and effective during deactivation), thereby potentially reducing the likelihood of flare-ups of inflammation and/or reducing the level of inflammation during flare-ups of inflammation or autoimmunity. ADCs may be preferred for use in the treatment of chronic autoimmune and inflammatory diseases. This is of therapeutic importance as many autoimmune/inflammatory diseases are in remission/relapse, and therefore there is an important clinical need for drugs and biologics used to treat such conditions. The aim is to provide a treatment regimen in which the disease is effectively managed both during remission and relapse so that the patient does not suffer from tissue damage.

Furthermore, of these ADC targets, only VISTA is expressed on neutrophils. This is because neutrophils are important during the early (acute) stages of inflammation, especially during bacterial infections, environmental exposures, and some cancers, and actually move towards the site of inflammation via chemotaxis. It is important because it is one of the first responders of migrating inflammatory cells. (Yoo SK et al., (November 2011). “Lyn is a redox sensor that mediates leukocyte wound attraction in vivo”. Nature. 480 (7375): 109 -12). Also, since these cells are expressed in the early stages of the inflammatory response, VISTA ADCs are expected to have a rapid onset of action (as indeed demonstrated herein).

Among its additional therapeutic importance, VISTA is also not expressed on B cells (unlike some other ADC targets, such as CD40 and CD74), so VISTA ADCs can be used to target B lymphocytes during treatment. should not affect Thus, VISTA ADCs may preserve humoral immunity during treatment, which may reduce the likelihood that a subject will develop an infection or even cancer during treatment. (Since steroids are powerful immunosuppressants, an associated risk, especially during chronic use, is that the treated subject may develop a fatal infection or malignancy during treatment).

Also, among these ADC targets, only VISTA appears to be constitutively expressed by naive Tregs, CD4 ⁺ T and CD8 ⁺ T cells. This is particularly important because these cells are involved in inflammatory responses and also because Tregs have recently been reported to be critical to the effectiveness of steroids. (Buttgereit, Frank and Timo Gaber, Timo, Cellular and Molecular Immunology, “New insights into the fascinating world of glucose ticoids: the dexamethasone-miR-342-Rector axis in regulatory T cells”, Vol. 18, 520-522 (2021) , and Immunity, “Anti-inflammatory Roles of Glucocorticoids Are Mediated by Foxp3+Regulation T Cells via a miR-342-Dependent Mec. hanism:, Vol. 53(2): 581-596 (September 2020), Braitch M. et al., Acta Neurol Scand., “Glucocorticoids increase CD4+CD25high cell percentage and Foxp3 expression in patients with multiple scleros is”, 2009 April; 119(4):239-245).

Indeed, the experimental evidence contained herein shows that the VISTA ADC effectively targets these different types of immune cells and is effective (i.e., therapeutic to different types of immune cells). Inflammation) provides internalization of the amount of steroids).

Example 19: Summary of PK vs. PD As mentioned above, the subject anti-inflammatory drug conjugates provide much longer PD duration than expected given the short PK of the anti-VISTA antibodies included in the conjugates. . PK, PD, and Kd values for exemplary anti-VISTA antibodies and ADCs included in accordance with the present invention are summarized in Table 7.

The CDR and variable sequences of the antibodies identified in Table 7 are found in FIGS. 8, 10, and 12. The PD or potency referring to FKBP5 in the table is defined as the 2-fold induction of FKBP5 in macrophages by PBS at 14 days after administration. PD or potency, which refers to "cytokine reduction" in the table, is defined as a 20% reduction in TNFα at 7 days post-dose in an ex vivo macrophage activation assay. These assays are illustrated in Example 6.

The data in Table 7 shows that the exemplary antibodies (which all bind to human VISTA-expressing immune cells at physiological pH and have short pKs) nevertheless provide long PDs, i.e., longer PDs than those of the therapeutic antibodies. (and more typical) as expected for antibodies with PK. This data indicates that the subject ADC should be suitable for uses where extended efficacy is desired.

Example 20: Anti-VISTA antibody drug conjugates have target-dependent effects in blood but not in tissues where there are few or no target-expressing cells.
These studies were conducted to assess the targeting specificity of antibody drug conjugates (ADCs) INX231P and INX234P to VISTA-expressing tissues. To monitor/confirm GC delivery and activity, the effects of genome-wide transcription via RNA sequencing (RNAseq) were investigated by Vermeer et al. (2003) Glucocorticoid-induced increase in lymphocytic FKBP51 messenger ribonucleic acid expression: a potential marker for glue cocorticoid sensitivity, potency, and bioavailability. J Clin Endocrinol Metab. Measured according to Jan;88(1):277-84. As shown in previous examples, INX201J results in rapid and long-term induction of FKBP5 in VISTA-expressing tissues such as the spleen. In contrast, non-VISTA expressing tissues showed little or no FKBP5 induction. Analyzes were performed in cynomolgus monkeys, comparing VISTA-expressing (blood) and non-expressing tissues (brain, white fat, and bone) (experiment 1), and in mouse bone (experiment 2).

As disclosed in previous examples herein, ADCs according to the present invention have been shown to be robust over long periods of time. In particular, we show that treatment with exemplary ADCs, namely INX231P and INX234P treatment, results in rapid and long-term (>4 days) induction of FKBP5, a direct transcriptional target of GCs, in VISTA-expressing tissues such as the spleen. In contrast, non-VISTA expressing tissues showed little or no FKBP5 induction.

This example demonstrates the target-specific effects of treatment with an ADC of the invention using RNAseq to assess global transcriptional changes. Analyzes were performed in non-human primates (NHP, cynomolgus monkeys) and compared transcription in VISTA-expressing (blood) and non-expressing tissues (brain, white fat, and bone) and mouse bone. In addition, intracellular accumulation of released payload (INX-SM-3), cysteine-modified linker payload (INXP-cys), and free dexamethasone was assessed across multiple VISTA-expressing and non-expressing tissues via mass spectrometry. Specifically, the experiments were carried out using a C57Bl/6 or human VISTA knock-in, in which the human VISTA cDNA was knocked in instead of the mouse VISTA gene, and expressing human VISTA at both the RNA and protein levels with the same expression pattern as mouse VISTA. (hVISTA KI) mice. Briefly, for mice, INX231P, PBS, or free dexamethasone (Dex) was delivered via intraperitoneal (i.p.) injection. After 20 hours for INX231P treatment and 2 hours for Dex treatment, bones were isolated, bone marrow flushed, and RNA extracted.

For NHP (cynomolgus monkey) studies, INX234P, vehicle, or free Dex was delivered intravenously (i.v.). Half of the animals in each group were sacrificed at 24 hours (excluding the vehicle group) and the other half after 7 days. At both time points, animals were bled and then underwent necropsy. Tissues were collected and RNA or proteins/peptides extracted.

For both studies, genome-wide transcript levels were assessed by RNAseq (Admera Health) and intracellular payload levels by mass spectrometry (Quintara Biosciences).

The purpose of these studies was to evaluate the effects of anti-VISTA steroid ADCs on gene expression in target-bearing and non-target-bearing tissues of both mice and NHPs. In addition, accumulation of released payload in target-bearing and non-target-bearing tissues in NHPs was evaluated via mass spectrometry (Quintara Biosciences).

Genome-wide changes in transcriptional expression were assessed via RNAseq (Admera Health). Transcripts specifically identified in the literature as being affected by glucocorticoid action were monitored. One of these transcripts is FKBP5, a sensitive and early GC response gene (Vermeer et al. (2003)). expression: a potential marker for glucocorticoid sensitivity, potency, and bioavailability.J Clin Endocrinol Metab. Jan;88(1):277-84)). We showed that Dex treatment causes a dramatic increase in FKBP5 messenger RNA in VISTA-expressing cells 2-4 hours after treatment, but that the transcriptional effects disappear by 24 hours (ADCINVIVO.04). In contrast, the effect of ADC on FKBP5 transcription is long-lasting, with peak induction at 20 hours post-treatment, although the signal is still detectable for 3 days in monocytes and 14 days in macrophages.

These experiments used anti-VISTA antibodies INX231 and INX234, each with an INX P linker payload described herein, delivered in vivo, or free Dex. Various tissues were collected and RNA isolation and transcriptional effects of treatment were evaluated for both mouse and cynomolgus tissue. Intracellular released payload and linker payload accumulation was assessed via mass spectrometry (Quintara Biosciences) for both Dex and INX234P treated groups across various cynomolgus monkey tissues. The materials and methods used are explained in particular detail below.

Materials and Methods Method Experiment 1
Two hours before mouse euthanasia and bone isolation, Dex was injected intraperitoneally, which corresponds to peak FKBP5 induction. Experiments were performed in C57Bl/6 female mice.

ADC (INX231P) was injected intraperitoneally 20 hours before euthanasia of mice to provide sufficient time for ADC treatment and peak potential gene transfer. Experiments were performed in hVISTA KI female mice.

A control group injected with PBS only was included to define the transcript baseline. After euthanasia, bones were isolated, bone marrow flushed, and bones were snap frozen in liquid nitrogen before RNA isolation and RNAseq analysis (Admera Health).

Vehicle, INX234P, or Dex was delivered intravenously to cynomolgus monkeys 24 hours before blood collection. Blood was sent to ImmuNext where it underwent red blood cell lysis, washed once with PBS, and cell pellets were then flash frozen in liquid nitrogen before RNA isolation. Monkeys were then euthanized and tissues perfused prior to tissue collection. Tissues were then again flash frozen in liquid nitrogen before RNA isolation and quantification via RNAseq analysis (Admera Health) or MS (Quintara Biosciences).

Materials Testing Drug and Dosage Antibody INX231P (lot number JZ-0556-017-1) has a drug/antibody ratio (DAR) of 8.0, being conjugated through full modification of interchain disulfides. It is INX231. The linker/payload (P) consists of a protease sensitive linker with a budesonide derivative payload.

INX234P (lot numbers JZ-0556-029, JZ-0556-017) is INX234, with a drug/antibody ratio (DAR) of 8.0, conjugated through full modification of interchain disulfides. . The linker/payload (P) consists of a protease sensitive linker with a budesonide derivative payload.

Dexamethasone For Experiment 1, Dex (sterile injection solution, Phoenix, NDC 57319-519-05) was diluted in PBS and administered as described via intraperitoneal injection. For Experiment 2, Dex was administered intravenously and supplied by Bimeda MTC.

Mice hVISTA KI mice were bred at the Center for Comparative Medicine and Research at Dartmouth, and C57Bl/6 mice were received from Jackson Laboratories (reference number 000665).

Female mice were enrolled between 9 and 15 weeks of age.

Cynomolgus Monkey Research was conducted at the Charles River Laboratory. Twelve female cynomolgus macaques were enrolled; they were purpose-bred, non-naïve, and ranged in age from 2 to 4 years.

Groups consisted of 2 animals in the vehicle control treatment group, 4 animals in the Dex treatment group, and 6 animals in the INX234P treatment group. Animals were injected on day 1.

All animals were bled at 24 hours and the remaining animals were bled again on day 8. Tissue analysis was performed on groups sacrificed at 24 hours (Dex and INX234P) and on day 8 (INX234P only), two animals treated with Dex and three animals treated with INX234P at 24 hours. Two animals injected with vehicle control, two animals treated with Dex, and three animals treated with INX234P were euthanized 8 days post-injection.

RNA preparation and RNAseq
Cell pellets from blood and brain were resuspended in 0.4 ml RNA lysis buffer from the NucleoSpin® RNA Plus kit (Macherey-Nagel, number 740984). RNA was isolated according to the manufacturer's instructions and eluted in 30 or 40 μl H2O (RNase/DNase free). RNA concentration was assessed on a Nanodrop.

RNA from bone and white adipose tissue was isolated by Admera Health. Quality control evaluation and RNAseq of all samples were performed by Admera Health.

Tissue Preparation for Mass Spectrometry Cell pellets from various tissues were homogenized in 2 volumes of 25% methanol. All samples were diluted with the corresponding plasma blank and each sample was extracted with acetonitrile containing an internal standard (verapamil). The mixture was vortexed on a shaker and then centrifuged. An aliquot of supernatant was transferred for injection into LC/MS/MS. Calibration standards and quality control samples were prepared by spiking test compounds into blank cynomolgus plasma and then processing with unknown samples.

Results Experiment 1
In this study, we evaluated the effects of INX231P and Dex on bone transcript levels in hVISTA KI (INX231P) versus C57Bl/6 mice (Dex/PBS). INX231P was administered at 10 mg/Kg (delivering a payload of 0.2 mg/Kg). Bones were isolated, bone marrow flushed, and transcript expression levels were measured 20 hours later, providing sufficient time for ADC treatment and potential peak transcript expression effects. Dex was injected at 2 mg/Kg, and transcriptional expression levels were measured 2 hours later. INX231P effects were measured 20 hours after a single intraperitoneal injection at 10 mg/Kg (delivering a payload of 0.2 mg/Kg). Dex effects were measured 2 hours after a single intraperitoneal injection at 2 mg/Kg. Gene transcript levels were measured by RNAseq and presented as normalized counts (n=5 mice/group, conventional one-way ANOVA compared to PBS only group).

As shown in the experiments in Figure 105, robust FKBP5 induction was observed with Dex treatment, but INX231P caused smaller changes in FKBP5 signaling, likely driven by VISTA-expressing immune cells. Similarly, Dex treatment had significant effects on several other osteotoxicity-related transcripts such as Rankl, Ddit4, Fam107a, and others (e.g., Stc2, Runx2, Errfl1 - not shown in data). . The results show that the ADC of the invention, INX231P, had no significant effect on the same transcripts. As shown in Figure 105, steroid responsive genes in bone are significantly affected by Dex. INX231P has limited effect. Fkpb5 (left) RANKL (center left) ddit4 (center right) Fam107a (right). INX231P effects were measured 20 hours after a single intraperitoneal injection at 10 mg/Kg (delivering a payload of 0.2 mg/Kg).

Experiment 2
RNAseq Analysis In this experiment in Figure 106, INX234P vs. The influence of Dex was evaluated. INX234P effects were measured 24 hours after a single intravenous dose at 10 mg/Kg (delivering a payload of 0.2 mg/Kg). Gene transcript levels were measured by RNAseq and presented as normalized counts (n=2 vehicles, n=6 ADCs/group, unpaired T-test vs. vehicle). The results show that in blood, treatment with an exemplary ADC according to the invention, INX234P, results in a distinct steroid signature with INX234P (FIG. 106). As shown therein, induction of steroid-responsive gene expression in peripheral blood cells of cynomolgus monkeys by ADC compared to vehicle controls.

As further shown in the experiments in Figure 107, a single intravenous dose at 10 mg/Kg (delivering a payload of 0.2 mg/Kg) inhibited the effects of INX234P in brain, white fat, and bone on steroid-related transcripts. Measurements were taken 24 hours later or on D8 (vehicle). Gene transcript levels were measured again by RNAseq and presented as normalized counts. (n=2 vehicle, n=3 ADC/group for tissue, unpaired T-test vs. vehicle-INX234P was not significant). The results in Figure 107 show that in brain, white fat, and bone, steroid-related transcripts at 24 hours showed limited changes or were similar to controls. There is also some within-group variation in these results, which is likely due to the small number of animals and heterogeneity between monkeys.

In the experiment of Figure 108, steroid-responsive genes appear to exhibit residual Dex effects at 24 hours in white adipose tissue. As shown, ADC gene expression is similar to vehicle control. In this experiment, the effects of free Dex (2 mg/Kg) and INX234P (10 mg/Kg, delivering a payload of 0.2 mg/Kg) were evaluated 24 hours or 8 days after a single intravenous administration (vehicle). was measured. Gene transcript levels were measured by RNAseq and presented as normalized counts. (n=2 vehicles, n=dexamethasone, for tissues n=3 ADCs/group, unpaired T-test vs. vehicle).

The results in Figure 108 show that at 24 hours, Dex had little or no residual effect on direct steroid responsive genes such as FKBP5, which correlates with rapid clearance. Of note, in white fat, PDE3B is an indirect marker of glucocorticoid action known to be downregulated by GCs. PDE3B was observed to be downregulated by Dex, but in ADC-treated monkeys, transcript levels were similar to controls (Figure 101) (Lee R. et al. (2018) Glucocorticoid receptor and adipocyte biology. Nucl Receptor Res. 5:101373.). Other direct targets of steroid action known to be upregulated by treatment (ANGPTL4, Mgll) also appeared to be downregulated at 24 hours in the Dex group (Figure 108) (Lee R. et al. (2018) Glucocorticoid receptor and adipocyte biology. Nucl Receptor Res. 5:101373.).

We hypothesize that this may be the result of a negative feedback loop after the initial upregulation, providing some confirmation of where transcriptional expression changes occur with glucocorticoid treatment.

MS Analysis In the experiments of Figures 109A-D, the concentrations of the released payload (INX-SM-3) and cysteine-modified linker payload ((INXP-cys) This experiment showed that high accumulation of active payload (INX-SM-3) at 24 hours in INX234P-treated monkeys correlated with VISTA-expressing tissues. (A) Released payload (INX-SM-3), or (B) cysteine-modified linker/payload (INXP-cys), or (C) dexamethasone with INX234P (10 mg/kg, delivering a payload of 0.2 mg/Kg) or free dexamethasone. (2 mg/kg). Accumulated compound levels were measured by LC-MS/MS and presented as ng compound/g tissue (n = 3 ADCs/group, n = 2 dexamethasone).

In particular, at 24 hours there is strong accumulation of released payload in VISTA-expressing immune tissues, with limited or no accumulation in non-VISTA-expressing tissues such as the colon and duodenum, adipose, or brain. (Figure 109A), and the payload was also detected in the liver and kidneys, organs responsible for filtration and excretion (Figure 109A). There was also some accumulation of cysteine-modified linker payload, the expected product of catabolic ADC prior to payload release (Figure 109B). As expected, Dex-treated monkeys accumulated little free dexamethasone in any tissue over 24 hours (Figure 109C). Residual dexamethasone was not correlated with VISTA expression, but was instead concentrated in liver, kidney, and white fat. In direct contrast to Dex, which was only present at low levels at 24 h, robust levels of the released payload (INX-SM-3) persisted at day 8 (end of study) in VISTA-expressing tissues. , with only low or undetectable levels in non-VISTA expressing tissues (Figure 109D).

Conclusions The results of Experiment 1 showed that Dex administered at 2 mg/Kg induced robust levels of Fkbp5, Rankl, Ddit4, and other steroid- and bone-specific toxin-related transcripts in mouse bone samples. Indicated. In contrast, INX231P administered at 10 mg/Kg (0.2 mg/kg payload) showed limited changes in the expression of the same transcripts compared to the untreated group. One of the major toxicities associated with long-term GC treatment is glucocorticoid-induced osteoporotic bone damage. These data argue that steroid anti-VISTA conjugates have limited bone-related toxicity.

The results of Experiment 2 showed that in female cynomolgus monkeys, INX234P administered at 10 mg/Kg (or 0.2 mg/Kg payload) significantly reduced blood FKBP5 and other steroid pathway-related transcripts compared to vehicle controls. It was shown that it had a strong impact on In tissues with little or no VISTA expression, transcript levels appeared to be more similar to controls, but results were influenced by substantial inter-animal variability and small sample numbers.

Dex at 2 mg/Kg had limited effect in most tissues considering that small molecules should be cleared in 24 hours. These findings correlated with robust intracellular accumulation of released payload from the INX234P-treated group at 24 hours in VISTA-expressing tissues and limited accumulation in non-VISTA-expressing tissues. After 7 days (study day 8), robust levels of released payload persisted in VISTA-expressing tissues such as lymph nodes (both ilium and jaw) and bone marrow. This is in stark contrast to free dexamethasone, which was detected at very low levels in non-immune tissues, as expected by 24 hours.

Interestingly, Dex at 2 mg/Kg had limited effects in most tissues, given that small molecules should be cleared in 24 hours, but some transcripts were negatively affected. potentially down-regulated due to a feedback loop. This provides an indication of transcripts that should indicate steroid effects, if present, and in particular have limited or no effect of anti-VISTA steroid ADCs.

The results also showed that the transcriptional data were strongly supported by the payload accumulation data. INX234P-treated monkeys showed robust levels of the released active payload of INX234P, INX-SM-3, and lower levels of the intact linker payload ((INXP-cys) in VISTA-expressing tissues. Low Levels of residual dexamethasone did not correlate with VISTA expressing tissues.

Specifically, data from Experiment 1 showed that in mouse bone samples, Dex administered at 2 mg/Kg resulted in robust levels of Fkbp5, along with additional osteotoxicity-associated transcripts such as Rankl, ddit4, and Fam107a, among others. Indicates that the In contrast, INX231P administered at 10 mg/Kg (0.2 mg/kg payload) showed very limited changes in the expression of the same transcripts or no changes compared to the untreated group. showed no change.

In female cynomolgus monkeys, INX234P administered at 10 mg/Kg (or 0.2 mg/Kg payload) had robust effects on FKBP5 and other steroid pathway-related transcripts in the blood compared to vehicle controls. Gave. In tissues with little or no VISTA expression, transcript levels appeared to be more similar to controls, but results were influenced by substantial inter-animal variability and small sample numbers.

Also, Dex at 2 mg/Kg had limited effects in most tissues, given that small molecules should be cleared in 24 hours, but some transcripts may be affected by negative feedback. Potentially downregulated for loops. This provides an indication of transcripts that should indicate steroid effects, if present, and in particular have limited or no effect of anti-VISTA steroid ADCs.

Transcriptional data were strongly supported by payload accumulation data. INX234P-treated monkeys showed robust levels of the active payload, INX-SM-3, and lower levels of the intact linker payload ((INXP-cys) released in VISTA-expressing tissues. Day 8 At (study end), robust levels of released payload persisted in VISTA-expressing tissues (lymph nodes and bone marrow). Low levels of residual dexamethasone at 24 hours did not correlate with VISTA-expressing tissues. Ta.

Example 21: Non-GLP pharmacokinetic study of INX234P by intravenous injection in cynomolgus monkeys After a single injection at 15 mg/Kg in cynomolgus monkeys, an exemplary anti-human VISTA monoclonal antibody conjugated to a glucocorticoid (GC) payload according to the invention Studies were conducted at Charles River laboratories (CRL) to determine the pharmacokinetic properties of a unique ADC, INX234P. Changes in immune populations, cortisol levels, and intracellular/serum levels of GC payloads were also measured.

In these experiments, changes in white blood cell (WBC) counts and cortisol levels were monitored over the course of the experiment. Accumulation of released payload was also quantified in both serum and blood cell pellets by mass spectrometry (MS).

Materials and Methods Experimental Design Four male cynomolgus monkeys received a single dose of INX234P at 15 mg/Kg intravenously (i.v.). Animals were then bled at the time points listed below. Serum and blood cell pellets were collected and stored at all time points.
Test drug and dosage INX234P (Abzena, HA-0853-02)
15 mg/Kg was administered intravenously.

Cynomolgus Monkeys Studies were conducted at CRL on purpose-bred, non-naïve, non-human primates from RMS Houston (Houston, TX, USA) and Orient BioResource Center (Alice, TX, USA).

Four males aged 2-4 years were enrolled in the study.

Details of sample collection are in Table 8.

Hematology Hematology details are in Table 9.

Blood smears were prepared from each hematological sample.

Bioanalytical Sample Processing PK blood samples were centrifuged and the resulting serum was separated and immediately frozen on dry ice or in a freezer set to maintain below -70°C.

Bioanalytical Sample Analysis 96-well polystyrene microplates were coated with 0.5 μg/mL coating solution (sheep anti-human IgG, The binding site, catalog number AU003.M) for 12-24 hours at 2°C-8°C. After washing, blocking buffer (Pierce/Thermo Scientific, cat. no. 37528) was added and incubated at room temperature on a plate shaker for 90 minutes ± 10 minutes at 350 rpm. Plates were washed, test samples were added, and incubated at room temperature on a plate shaker for 2 hours ± 20 minutes at 350 rpm. Wash the plate and add 250 ng/mL detection antibody solution (Goat anti-human IgG, HRP (H&L), Bethyl, Cat. No. A80-319P) to each well of the plate(s) and incubate on a plate shaker at room temperature. The cells were incubated at 350 rpm for 60 hours ± 5 minutes. The plates were washed and 100 μL of TMB substrate solution was added to each well(s) of the plate(s) and incubated for 15±2 minutes at room temperature. The TMB reaction was stopped by adding 100 μL of stop solution to each well of the plate(s).

Plates were read at 450 nm within 15 minutes of stopping the reaction using a microplate reader (Molecular Devices SPECTRAmax). The test articles were used in the calibration curve.

Pharmacokinetic evaluation Antibody half-life was determined using the PKsolver program with non-compartmental analysis (NCA) after an intravenous bolus.
cortisol ELISA
Cortisol enzyme immunoassay kit (Arbor Assays, catalog number K003-H5).
Serum samples were assayed using a cortisol ELISA kit according to the supplier's protocol.

Mass spectrometry MS quantification was performed on cell pellets and serum by Quintara Discovery.

On the cell pellet:
Briefly, cell pellets were suspended in 100 μL of 20% methanol. All samples were diluted with the corresponding plasma blank and each sample was extracted with acetonitrile:methanol containing an internal standard (verapamil). The mixture was vortexed on a shaker and then centrifuged. An aliquot of supernatant was transferred for injection into LC/MS/MS (ExionLC AD pump, Sciex Qtrap 6500+). Calibration standards and quality control samples were prepared by spiking test compounds into blank cynomolgus plasma and then processing with unknown samples.

Normalization of data to cell volume (ng/ml) was performed by first calculating cell number based on a fixed volume of each sample processed for PBL or WBC hematology counts and MS. . It was then converted to volume by using the most frequently employed arbitrary mean corpuscular volume (MCV) of 400 femtoliters per cell (3).

On serum:
Each plasma sample was extracted with acetonitrile:methanol containing an internal standard (verapamil). The mixture was vortexed on a shaker and then centrifuged. An aliquot of supernatant was transferred for injection into LC/MS/MS (ExionLC AD pump, Sciex Qtrap 6500+). Calibration standards and quality control samples were prepared by spiking test compounds into blank cynomolgus plasma and then processing with unknown samples.

All graphs were created with GraphPad (Prism).

Results Pharmacokinetic Data Animals received a single dose of INX234P intravenously at a dose of 15 mg/Kg, and PK data was analyzed using PK Solver, resulting in PK or T1/2 = 14 hours.

The experiment in Figure 111 shows INX234P PK analysis. In the experiment, INX234P was administered intravenously at 15 mg/Kg, animals were bled at the indicated time points, serum was isolated, and antibody levels were measured (n=4 cynomolgus monkeys, SEM).

Additionally, raw PK data is included in Tables 10 and 11 below.

Hematological Changes As shown by the results of the experiment in Figure 112, all animals showed a general increase in white blood cells (WBC), with numbers returning to normal by 48 hours post-injection. Lymphocytes (LYMPH) showed a rapid decrease 3 hours after injection. The numbers returned to normal by 96 hours. Monocytes (MONO) showed a decrease by 24 hours post-dose, but returned to normal by 96 hours. These changes are consistent with the normal effects of GC, which is known to cause transient leukopenia.

More specifically, the results in Figure 112 show the hematological changes induced by a single dose of INX234P. INX234P was administered intravenously at 15 mg/Kg. Animals were bled at the indicated time points, blood smears were performed, and different cell populations were counted (n=4 cynomolgus monkeys, SEM) (WBC=white blood cells, NEUT=neutrophils, LYMPH=lymphocytes, MONO=monocytes). globules, EOS = eosinophils, BASO = basophils, RBC = red blood cells, Retic = reticulocytes).

Cortisol Changes Basal cortisol levels were highly variable between animals (193.7 ng/ml to 298.8 ng/ml). Twenty minutes after intravenous injection, all animals showed stress-induced increases in cortisol. By 12 hours post-injection, all animals had cortisol levels falling back to normal with high inter-animal variability, but returned to normal by 24 hours (experiment in Figure 113). Considering that time 0 = 8 a.m., the decrease in cortisol at approximately 12 hours = 10 p.m. is consistent with normal circadian rhythms in cynomolgus monkeys (Tochitani et al, 2019, “Physiological and drug-induced changes in blood levels of adrenal steroids and their precursors in cynomolgus monkeys: An application of steroid profiling by LC-MS/MS for evaluation of the adrenal toxicity”, Toxicol. Sci. Vol. 44, No. 9, 575-584).

More specifically, the results in Figure 113 show cortisol changes induced by a single dose of INX234P in individual animals. INX234P was administered intravenously at 15 mg/Kg. Animals were bled at the indicated time points, serum was isolated, and cortisol levels were measured by ELISA (n=4 cynomolgus monkeys).

Payload Accumulation in Serum and Peripheral Blood Leukocytes Previous experiments showed that the major metabolite or released payload of INX234P is INX-SM-3. Both serum and peripheral blood leukocytes (PBL) were sent to Quintara Discovery to perform MS analysis for quantification of linker payload (P-cys) and released payload (SM3) over time.

The experiment in Figure 114 shows that by an early time point, the conjugate is internalized, the antibody begins to be catabolized, and a portion of the complete linker payload is released (INX P-cys). This accumulates intracellularly and is then cleaved, resulting in intracellular concentrations of the active released payload (INX-SM-3).

More specifically, the experiment in Figure 114 shows the PK of linker payload (P-cys) and released payload (SM3) in PBL when INX234P was administered intravenously at 15 mg/Kg. Animals were bled at the indicated time points and PBLs were isolated and analyzed by MS (n=4 cynomolgus monkeys, SEM). Since the conjugate is plasma stable for 7 days (data not shown), the released payload and linker payload in the plasma represents payload efflux from the cells.

The experiment of FIG. 115 shows that in serum, P-cys peaks at about 12 hours, while maximum concentration for released payload SM3 is reached at about 24 hours. Of note, intracellular levels are 100-fold higher for linker payloads and 20-fold for released payloads compared to serum, which allows efficacy but limits serum exposure of non-targeted tissues. expensive.

By day 8 (192 hours) post-injection, both released payload and linker payload remain undetectable despite the presence of substantial tissue levels of released payload in lymph nodes and bone marrow. Below. More specifically, the experiment in Figure 115 demonstrated the linker payload in the serum when INX234P was administered intravenously at 15 mg/Kg, the animals were bled at the indicated time points, the serum was isolated, and the linker payload in the serum was analyzed by MS. (P-cys) and the PK of the released payload (SM3).

INX234P induced transcriptional changes over time in peripheral blood leukocytes. The experiment shown in Figure 116 used blood collected before INX234P administration (or before blood collection) as a baseline level (to calculate fold changes). ) shows the transcriptional changes in PBL evaluated during the course of the PK study.

As shown in Figure 116, despite inter-animal variation, steroid transcriptional targets such as FKBP5, DUSP1, ZBTB16, TSC22D3, and NFKBIA showed a rapid increase in transcription as early as 3 hours post-administration, and FKBP5 , DUSP1, and ZBTB16 peak at about 12 hours. At 24 hours, most transcriptional targets are still upregulated.

As shown therein, at 96 hours, the transcript levels of FKBP5, ZBTB16, and NKBIA were still 2-fold higher than baseline, while DUSP1, SGK1, and TSC22D3 had returned to baseline.

More specifically, the experiment in Figure 116 shows that steroid responsive genes in PBL are upregulated by INX234P when INX234P is administered intravenously at 15 mg/Kg. Animals are bled at the indicated time points, PBLs are isolated, subjected to RNA isolation, and gene transcript levels are measured by RNAseq and presented as fold change versus pre-bleeding (n=4 cynomolgus monkeys).

Conclusion The data for this example shows the following.
-INX234P has a half-life (T _1/2 ) of 14 hours.
-INX234P induces limited and transient cellular changes in peripheral blood, as expected for GC. The antibody component does not appear to add toxicity.
- No consistent changes in cortisol levels were observed after INX234P administration, except as expected due to circadian rhythms.
- Released linker payload and subsequent release payload accumulation in blood cells occurs within a few hours, resulting in intracellular concentrations in blood at levels 20-100 times higher than in serum.
- There are no detectable levels of PBL at day 8, although the released payload still persists in the bone marrow and lymph node tissues, probably due to cell turnover.
-INX234P induces transcriptional changes in PBL as early as 3 hours post-dose, with some targeted changes still detectable at 96 hours, indicating that the delivered payload is functional with an extended pharmacodynamic range. indicates that it is active.
1-Vermeer et al. (2003) Glucocorticoid-induced increase in lymphocytic FKBP51 messenger ribonucleic acid expression: a potential marker for glue cocorticoid sensitivity, potency, and bioavailability. J Clin Endocrinol Metab. Jan;88(1):277-84
2-McPherson MJ, et al. (2017) Glucocorticoid receptor agonist and immunoconjugates thereof, US15/611,037
3-Khoo et al, (2002) Intracellular Accumulation of Human Immunodeficiency Virus Protease Inhibitors, Antimicrob. Agents Chemother. 2002 Oct; 46(10):3228-3235.
4-Tochitani et al, 2019, J. Physiological and drug-induced changes in blood levels of adrenal steroids and their precursors in cynomolgus monkeys: An ap plication of steroid profiling by LC-MS/MS for evaluation of the adrenal toxicity, Toxicol. Sci. Vol. 44, No. 9,575-584

Example 22: Identification and Quantification of Released Payload of INX Antibody Glucocorticoid Conjugates in Human Peripheral Blood Mononuclear Cells In this example, experiments were conducted to demonstrate that an exemplary anti-VISTA glucocorticoid conjugate according to the present invention , was shown to be rapidly internalized by VISTA-expressing cells. The antibody is catabolized releasing the cysteine-modified complete linker payload. The linker payload is also cleaved releasing the active payload. Given the diversity of proteases within cells, the released payload must be confirmed experimentally. In this study, we identified released payloads from internalized novel antibody glucocorticoid conjugates in human peripheral blood mononuclear cells (PBMCs) and determined their abundance via mass spectrometry (MS) for three different glucocorticoid linker payloads. was quantified.

The aim of this study was to identify and quantify the released payload of the novel glucocorticoid conjugates according to the invention in human PBMCs.

Materials and Methods Experimental Design In all experiments below, human PBMCs isolated from one healthy donor per study were treated with exemplary anti-VISTA glucocorticoid conjugates according to the present invention and released. This enabled identification and quantification of the payload.

Pilot experiments showed that 4 hour (h) incubation with anti-VISTA glucocorticoid conjugate resulted in a robust increase in FKBP5 in human PBMC. This is a direct effect of glucocorticoid activity.

In this experiment, PBMCs were incubated with glucocorticoid (GC) antibody-drug conjugates (ADCs) for 4 hours, after which cells were harvested and evaluated for both FKBP5 transcriptional induction and release via quantitative real-time PCR (qPCR). The resulting payload was evaluated via mass spectrometry (MS) for identification and quantification.
Reagent test ADC
- INX201J (Abzena, lot number JZ-0556-025-1) is conjugated to a linker/payload via full modification of interchain disulfides, with a drug/antibody ratio (DAR) of 8.0. It is INX201. The linker/payload (J) is based on the patent reported linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker with a budesonide analogue payload (INX J-2).
• INX231J (Abzena, lot number JZ-0556-013-1) is a conjugated INX231 with a DAR of 8.0. The linker/payload (J) is based on the patent reported linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker with a budesonide analogue payload (INX J-2).
• INX231P (Abzena, lot number JZ-0556-017-1) is INX231, with a DAR of 8.0, conjugated through full modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-3).
• INX231V (Abzena, lot number PP-0920-014-2) is INX231, with a DAR of 7.8, conjugated via interchain disulfide modification. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-32).
• INX234A11 (Abzena, lot number RJS-1054-001) is an INX234 antibody with a DAR of 8.0 that is conjugated through full interchain disulfide modification. The linker/payload (INX A11) consists of a negatively charged protease sensitive linker (Asn/gly) with a budesonide analog payload (INX-SM-32).
• INX234A3 (Abzena, lot number PP-0924-023-1) is an INX234 antibody with a DAR of 8.0 that is conjugated through full interchain disulfide modification. The linker/payload (INX A3) consists of a positively charged protease-sensitive linker with a budesonide analog payload (INX-SM-32).
• INX234V (Abzena, lot number RJS-1054-003) is an INX234 antibody with a DAR of 8.0 that is conjugated through full modification of interchain disulfides. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-32).
• INX231V (Abzena, lot number PP-0920-014-2) is INX231, with a DAR of 7.8, conjugated via interchain disulfide modification. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-32).
• INX231A7 (Abzena, lot number RJS-1054-007-001) is an INX231 antibody with a DAR of 7.8 that is conjugated via interchain disulfide modification. The linker/payload (INX A7) consists of a negatively charged protease-sensitive linker with a budesonide analogue payload (INX-SM-32) that is phosphorylated.
• INX231A12 (Abzena, lot number RJS-1054-007-002) is an INX231 antibody with a DAR of 6.99 that is conjugated via interchain disulfide modification. The linker/payload (INX A12) consists of a negatively charged protease-sensitive linker with the fluocinolone acetonide analog payload (INX-SM-25) being phosphorylated.
• INX231A23 (Abzena, lot number RJS-1054-006-001) is an INX231 antibody with a DAR of 7.34 that is conjugated via interchain disulfide modification. The linker/payload (INX A23) consists of a negatively charged protease-sensitive linker with a fluocinolone acetonide analog payload (INX-SM-25).
• INX234A5 (Abzena, lot number RJS-1054-002) is an INX234 antibody with a DAR of 7.9 that is conjugated via interchain disulfide modification. The linker/payload (INX A5) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-44).
• INX234A4 (Abzena, lot number PP-0924-023-2) is an INX234 antibody with a DAR of 7.9 that is conjugated via interchain disulfide modification. The linker/payload (INX A4) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-43).
• INX231J (Abzena, lot number JZ-0556-013-1) is an INX231 antibody with a DAR of 8.0 that is conjugated via interchain disulfide modification. The linker/payload (INX J) is based on the patent reported linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker with a budesonide analogue payload (INX J-2).
• INX231P (Abzena, lot number JZ-0556-017-1) is INX231, with a DAR of 8.0, conjugated through full modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-3).
• INX234P (Abzena, lot number HA-0853-02) is INX234, with a DAR of 8.0, conjugated through full modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-3).
• INX234J (Abzena, lot number JZ-0556-013-2) is an INX234 antibody with a DAR of 8.0 that is conjugated through full modification of interchain disulfides. The linker/payload (INX J) is based on the patent reported linker/payload (US 15/611,037). It consists of a negatively charged protease-sensitive linker with a budesonide analogue payload (INX J2).
• INX231S (Abzena, lot number PP-0924-014-1) is an INX231 antibody with a DAR of 6.9 that is conjugated via interchain disulfide modification. The linker/payload (INX S) consists of a negatively charged protease-sensitive linker with a fluocinolone acetonide analog payload (INX-SM-24).
Cell culture medium ●RPMI 1640 (VWR, catalog number 16750-084) without L-glutamine
●Penicillin/Streptomycin/Glutamine (ThermoFisher, catalog number 10378016)
●1M Hepes (Gibco, catalog number 15630-080)
●Human AB serum (Valley Biomedical, catalog number HP1022HI)
Other reagents Ficoll-Paque Plus (GE Healthcare, catalog number 17-1440-03)
●Histopaque 1077 (Sigma Aldrich)

PBMC Preparation Human PBMC were isolated under sterile conditions from apheresis cones obtained from the blood donor program at Dartmouth Hitchcock Medical Center from de-identified healthy human donors.

Blood was transferred to a 50 ml Falcon tube and diluted to 30 ml with PBS. 13 ml of Histopaque 1077 was slowly layered under the blood and the tube was centrifuged at 850 x g for 20 min at room temperature, with gentle acceleration and without interruption.

Mononuclear cells were collected from the plasma/Ficoll interface, resuspended in 50 ml of PBS, and centrifuged at 300 xg for 5 minutes. Cells were resuspended in PBS and counted.

Assay protocol for both metabolite ID and quantification Isolated PBMC were resuspended in RPMI 1640 (assay medium) containing 10% human AB serum, 10 mM Hepes, 1× Penicillin/Streptomycin/L-Glutamine.

PBMCs from one donor were washed twice in PBS and then resuspended in medium at 1 x 10^7/mL. 1 mL was transferred to each well of the 6-well plate used.

ADC or medium alone was added to each well of a six-well plate used for a final concentration of 1 mM payload with a total volume of 2 mL per well after drug addition. Plates were placed at 37°C for 4 hours.

After 4 hours, the medium containing cells was harvested and combined from all wells of a given treatment.

1 mL of the cell suspension was pelleted, the medium was removed, and the pellet was resuspended in RNA lysis buffer to enable FKBP5 analysis.

The remaining cells from each treatment were divided into aliquots representing 50 million cells per aliquot (for metabolite quantification, only one aliquot per condition was generated).

Cells were pelleted at 450 rcf for 4 minutes.

The medium was removed from each cell pellet containing medium and then set aside at -80°C until it could be assessed for the concentration of effluxed payload. Any additional residual medium was removed from the tube, leaving a dry cell pellet that was stored at -80°C for MS analysis.

As an additional control, media and an aliquot of stock drug (2mM) were diluted to final concentration (1mM) to confirm lack of free payload in the media at time 0.

MS analysis MS analysis was performed by Quintara Discovery.

For Metabolite ID Briefly, pre-chilled 70% ACN (extraction buffer) was added to the PBMC pellet for extraction. The pellet was suspended in extraction buffer and incubated in an ice bath. The samples were then centrifuged and the supernatant was collected from each sample. The supernatant was dried by Speed Vac and reconstituted with 50% MeOH. 15 mL was injected into LCMS (Luna C18(2) column, Thermo Scientific Q Exactive Hybrid Quadrupole Orbitrap) and the resulting mass was evaluated.

For quantification Briefly, cell pellets were suspended in 100 μL of 20% methanol. Each sample was extracted with acetonitrile:methanol (95:5, v/v) containing an internal standard (verapamil). The mixture was vortexed on a shaker and then centrifuged. An aliquot of supernatant was transferred for injection into LC/MS/MS (Sciex Qtrap 6500+). Test compounds were spiked into untreated human PBMC, calibration standards and quality control samples were prepared, and then treated with unknown samples.

RNA preparation and real-time PCR
The cell pellet was resuspended in 0.4 ml RNA lysis buffer from the NucleoSpin® RNA Plus kit (Macherey-Nagel, number 740984). RNA was isolated according to the manufacturer's instructions and eluted in 30 or 40 ml H2O (RNase/DNase free). RNA concentration was assessed on a Nanodrop.

Reverse transcription was performed using Taqman reverse transcription reagents (number N8080234) and according to the manufacturer's instructions.

Quantitative real-time PCR was performed using the Taqman Master Mix 2X kit (number 4369016) and run on a QuantStudio3 from Applied Biosystem. Primer used:
i. TaqMan gene expression assay (FAM-MGB); Assay ID: Hs01561006_m1 (FKBP5)
ii. TaqMan gene expression assay (FAM-MGB); Assay ID: Hs01922876_u1 (GapDH)

Ct data were converted to ΔCt and ΔΔCt or Log2 fold change was compared to untreated controls.

Results Experiment 1
INX-SM-J2, INX-SM-3, and INX-SM-32 are the major metabolites of INX201J, INX231P, and INX231V, respectively.
This experiment identified the major metabolites from incubation of INX201J, INX231P, and INX231V after 4 hours of in vitro incubation with human PBMC (single donor).

As shown in Figure 117, incubation with all conjugates results in a robust increase in FKBP5 transcription. MS analysis confirmed that the major metabolites of either of the conjugates were their respective predicted released payloads; for INX201J, this corresponds to INX-SM-J2; INX231P and INX231V are compatible with INX-SM-3 and INX-SM-32, respectively. Each of these released payloads in purified form has been shown to be active and induce similar efficacy in other experimental results reported herein. For each of the three conjugates, there was also a lower level of uncleaved cysteine-modified linker payload representing the catabolic antibody.

The data in Table 12 provides the approximate abundance of released payload, uncleaved cysteine-modified linker payload, and compound in the medium representing the flux of released compound. Notably, the abundance of ejected payload from INX231V far exceeds the abundance of ejected payload from INX231P and INX201J.

Differences in the released payload between INX231P and INX201J indicate that INX201 is internalized much more rapidly both in vivo and in vitro than INX231 (data not shown), resulting in additional accumulation of INX201 and that This is overridden by the fact that the method results in a more ejected INX J payload than an INX P payload. Even when compared to more rapidly internalizing antibody conjugates, the level of released payload of INX V still substantially exceeds that of the corresponding INX J.

As shown in Table 12, there was a substantial release of INX V payload versus INX P or INX J. Intracellular payload abundance was estimated at 4 hours for human PBMCs incubated with 1 mM conjugate payload (approximately 20 mg/mL ADC) and quantified via MS analysis. The amount of effluxed payload present in the medium at 4 hours is also shown. Levels in the culture medium were parallel to samples from another experiment (n = 1 donor (ND - not detectable (estimated LLOQ = 0.1 ng); BQL - limit of quantification (<0.5 ng/mL)). is quantified rather than estimated.

Experiment 2
INX V released a payload that was present at a higher level than the released payload of INX P or INX J.
In this study, we determined the abundance of released payloads after 4-hour incubation of INX231J, INX231P, and INX231V with human PBMCs. Table 13 provides the released payload, cysteine modified linker payload, and compound abundance in the medium representing the flux of released compound. Also included as a control is the level of released payload detected when the ADC stock solution was diluted in medium (no cells present) and submitted for MS analysis.

The results of this study confirm and quantify a dramatic 235-fold enhancement (ng/ng) of INX V's released payload over INX P and even over INX J's released payload. This substantial enhancement of release was not expected based on the structural differences between the three linker payloads, and because the same antibody (INX231) and gly glu linker were used for all three conjugates, there was no contribution from the antibody or linker. It wasn't the result.

Despite the fact that the antibodies are the same as the INX P and INX J conjugates, the released payload of INX P is approximately three times the amount of released payload of INX J in 4 hours. These results further support the efficacy benefits observed in the LPS stimulated PBMC assay shown in the previous example.

The data in Table 13 shows that the level of ejected payload of INX231V exceeds that of INX231J or INX231P. The quantified intracellular payload abundance at 4 hours is shown therein for human PBMCs incubated with 1 mM conjugate payload (approximately 20 mg/mL ADC) and quantified via MS analysis. The amount of effluxed payload present in the medium at 4 hours is also shown. The released payload level detected in the stock solution was 0.8 ng/mL for INX231J or below the limit of quantification -<0.5 ng/mL (INX231V/INX231P). Cysteine quench payload levels in the stock solution were 0.9 ng/mL for INX231P or below the limit of quantitation <0.5 ng/mL (INX231V/INX231P).

Experiment 3
Quantification of released payload from INX A11, INX A3, INX V, INX A7, INX A12, INX A23, INX A5, INX A4, INX P, INX S, and INX J anti-VISTA conjugates incubated with human PBMCs. In this study, we determined the abundance of released payload from a series of novel glucocorticoid conjugates after 4 hours of incubation with human PBMC (one donor). Table 14 provides the released payload and compound abundance in the medium representing the flux of released compounds. This study also included, as a negative control, the level of released payload detected when the ADC stock solution was diluted with medium (no cells present) and submitted for MS analysis. All amounts are given in ng/mL. In two cases (INX234P and INX234J), some of the material was lost due to technical changes prior to analysis, so the amount of intracellular material is normalized to constitute the loss of material.

This study confirms and quantifies the dramatic enhancement (ng/ng) of the ImmuNext novel linker payload over INX J literature comparisons. Remarkably, when the antibody backbone was held constant (e.g., INX231) and conjugated to either INX J or ImmuNext linker payload, a greater amount of released ImmuNext payload was detected intracellularly. be done. In one case where they are more similar (e.g., INX234P vs. INX234J), INX234P was detected at higher levels (approximately 1.5 times) in the cells, but still in contrast to INX J. There was no detectable spilled payload in the medium. This is important because in vivo, spilled payloads can result in shortened retention by targeted cell types and non-specific uptake by other cells.

The data in Table 14 shows that the level of new linker payload released payload exceeds that of the INX J comparison. The quantified intracellular payload abundance at 4 hours is shown in the table for human PBMCs incubated with 500 nM conjugate payload (approximately 10 mg/mL ADC) and quantified via MS analysis. The amount of effluxed payload present in the medium at 4 hours is also shown. All released payload levels detected in the stock solutions were at BQL (below the limit of quantitation of <0.5 ng/mL).
Conclusions - Anti-VISTA conjugates with each of the specific linker payloads (INX J, INX V, and INX P) are each cleaved and release theorized released payloads. Additional alternative cleavage products are not present at detectable levels.
● Some residual fully cysteine modified linker payload is still present.
- The released payload from the INX V conjugate and other novel glucocorticoid linker payloads are in higher abundance than the corresponding released payload of INX J.

In particular, in this example experiment, the major metabolites of INX201J, INX231P, and INX231V were theorized to be the released payloads INX-J2, INX-SM-3, and INX-SM-32, respectively. Identified.

Surprisingly, the released payload of INX231V was present in substantially higher amounts than the payloads of INX P and INX J after 4 hours of incubation. Initial studies estimated >75 times greater abundance (ng/ng) for the payload of INX P and INX J conjugates. A second study confirmed that it was present at >230 times the abundance (ng/ng) relative to INX P, and even higher in the released payload of INX J.

The INX P conjugate also showed enhanced accumulation of released payload (approximately 3-fold) over the INX J conjugate when the same antibody was used for each conjugate.

This increased abundance has a substantial impact on both potency and PD. Additional studies using the delayed lipopolysaccharide (LPS) assay disclosed in the previous example showed that although the released payloads for the three were similarly potent in their free form, The conjugates have proven to be substantially more potent in their conjugated form than INX P, which is more potent than the INX J conjugate of the same antibody. The large intracellular depot of released payload observed here in vitro contributes directly to the sustained effects of several weeks observed in vivo, even though ADCs have a short serum half-life of only a few hours. obtain.

All additional novel glucocorticoid conjugates tested also showed enhanced or dramatically enhanced intracellular accumulation over INX J conjugated to the same antibody backbone, with no release leakage into the culture medium. There was very little payload. This data strongly supports the enhanced in vitro potency and long PD of the ADCs of the present invention compared to the expected potency considering the potency of the free payload form and the typically very short PD of small glucocorticoid molecules. .

Example 23: IBD or Colitis Studies The Dextran Sulfate Sodium Colitis Mouse Model (DSS) model is commonly used to evaluate potential IBD or colitis therapeutics. (Eichele et al., “Dextran sodium sulfate colitis murine model: An indispensable tool for advancing our understanding of inflammation. 23(33):6016-6029”). , used this animal model to preliminarily evaluate the efficacy of the ADC according to the invention for the treatment of colitis or IBD.

Also, as is commonly known, IBD and colitis are chronic conditions that are difficult to treat and manage effectively and can lead to sepsis and death if treated ineffectively. obtain. Currently, the main means of managing IBD or colitis disease involves chronic steroid administration. Unfortunately, however, this can potentially cause toxicity, e.g. due to steroid effects on non-targets (e.g. epithelial cells) and/or long-term immunosuppression.

In this preliminary experiment, one group of animals received a Dex ADC according to the invention (INX234P) at a steroid dose of 0.2 mpk every other day, and a second group of positive control animals received free Dex steroids at a steroid dose of 2 mpk. and a third negative control group of animals was untreated. There were 10 animals in each group. When animals started showing weight loss (day 7) (DSS started on day 0), ADC or Dex treatment was started. The experiment ended on day 13 when one group (dex treatment) reached the maximum allowable weight loss.

Results (preliminary, not shown) suggest that ADC demonstrated efficacy compared to untreated controls. The results also suggest that ADC did not induce the same toxicity as observed in free steroid-treated animals. In that regard, dexamethasone has been reported to cause toxicity in this IBD model, van Meeteren ME, Meijssen MAC, Zijlstra FJ. “The effect of dexamethasone treatment on murine colitis”, Scand J Gastroenterol 2000;35:517-521, and Ocon et al. , “The glucocorticoid budesonide has protective and deleterious effects in experimental colitis in mice”, Biochemical Pharmac. 116 (2016) 73-88).

Although these results are preliminary, they suggest that the subject ADCs may be useful in the treatment of colitis or IBD indications. They also suggest that the subject ADCs may be preferable to existing free steroid therapies to treat these chronic diseases, as they may alleviate toxicity that can occur during long-term free steroid therapy. are doing. This is further supported by Example 25.

Example 24: Effect of Antibody Drug Conjugates INX234P and INX234V on T Cell Proliferation and Survival in a Xenograft GvHD Model-2 Study A humanized mouse model of xenograft-versus-host disease (xenograft GvHD) is specific for human drug targets in vivo. enables the study of immunomodulatory compounds. These are based on immunodeficient strains of mice injected with human-derived peripheral blood mononuclear cells (PBMCs). The NOD-scid IL-2Rγ null (NSG) line lacks mature T cells, B cells, and natural killer cells and is amenable to heterologous GvHD studies.

In the NSG model of heterologous GvHD, donor human T cells are transplanted intravenously (i.v.) and allowed to proliferate robustly over time in recipient mice, resulting in anti-host cell reactivity leading to skin tissue infiltration. Mice lose weight and die from GvHD if left untreated. The time frame for disease progression can range from 3 to 6 weeks. The time of disease onset and progression can be accelerated by irradiating mice with 2.5-3 Gy before transplantation of human PBMC. In this case, disease begins after about 1-2 weeks and mice die by the 2-3 week mark.

Briefly, mice were first injected with 2.5 x ¹⁰⁶ human PBMCs along with a dose of control human IgG1, INX234, or INX234P (experiment 1) or INX234V (experiment 2). Disease progression was monitored by periodically weighing mice and measuring human T cell proliferation by absolute white blood cell counts (ALC) on peripheral blood. The aim of the study was to evaluate the ability of human VISTA antibody INX234 conjugated to two different GC payloads to influence the progression of heterologous GvHD.

Materials and Methods Study Design In these experiments, an initial dose of antibody or ADC was injected intravenously with donor cells, followed by treatment by intraperitoneal injection (i.p. p.).

Blood was collected only once on day 21 for experiment 1. Blood was collected once a week starting on day 15 for experiment 2.
Test drug and dosage INX234 (ATUM, lot number 72931.2.a) is a humanized anti-human VISTA antibody on a human IgG1/kappa backbone with L234A/L235A/E269R/K322A silencing mutations in the Fc region. .
• INX234P (Abzena, lot number JZ-0556-017-2) is INX234, with a drug/antibody ratio (DAR) of 8.0, conjugated through full modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-3).
• INX234V (Abzena, lot number RJS-1054-003) is an INX234 antibody with a DAR of 7.9 that is conjugated via interchain disulfide modification. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-32).
• huIgG1si (Aragen, lot number BP-2211-018-6) is an anti-RSV monoclonal Ab on a human IgG1/kappa backbone with an E269R/K322A silencing mutation in the Fc region.

All antibodies were diluted in PBS and injected intraperitoneally in a volume of 0.2 ml to deliver a dose of 10 mg/Kg.

In experiment 2, animals were treated with INX234V until day 27 and then treated with INX234P for the remainder of the experiment.

Mice Eight-week-old NSG ^mice were purchased from Jackson Laboratory ( ^NOD . They were kept under conditions that did not include their bodies. Mice were tattooed before the start of the experiment.

In experiment 1, mice were irradiated with 2.5 Gy 7 days before PBMC intravenous transplantation.

In Experiment 2, mice were not irradiated.

Blood Sampling and Immunostaining Peripheral blood was collected from the retroorbital cavity using a glass Pasteur pipette that was first rinsed with heparin to prevent clotting.

The single wash protocol described below allows for absolute blood cell counts.

10 ml of antibody cocktail (see right) was added directly to 100 ml of blood. After 30 minutes of incubation at room temperature (RT), 600 ml of BD FACS lysis buffer was added to the samples. After incubation for 30 minutes at room temperature, samples were spun at 550 rcf for 5 minutes, washed once in PBS, and then resuspended in a fixed volume of PBS. The entire sample was run on a MacsQuant flow cytometer to obtain absolute cell counts.

Peripheral Blood Mononuclear Cell Isolation Human PBMC were isolated under sterile conditions from apheresis cones obtained from the blood donor program at DHMC from de-identified healthy human donors. Blood was transferred to a 50 ml Falcon tube and diluted to 30 ml with PBS. 13 ml of Histopaque 1077 (Sigma Aldrich) was slowly layered under the blood and the tube was centrifuged at 850 rcf for 20 min at room temperature without interruption at gentle acceleration.

Mononuclear cells were then collected from the plasma/Ficoll interface, resuspended in 50 ml of PBS, and centrifuged at 300 xg for 5 minutes. Cells were resuspended in PBS and counted.

Disease Monitoring Mice were weighed three times per week and euthanized when body weight fell below 75% of initial body weight, as allowed by the IACUC organization.

Results Experiment 1
INX234P Treatment Prevents Human PBMC Proliferation To monitor heterologous GvHD progression, human T cell proliferation and weight loss were measured. As shown in Figure 119, the number of human PBMCs was significantly reduced in the INX234P treated group at day 21 after cell transplantation. In these experiments, peripheral blood was collected on day 21 and human CD45-positive cells were quantified by flow cytometry. Mice were dosed once a week from day 0 to day 34 (SEM, n=8/group) (dosing at 10 mg/Kg, INX234P provided 0.2 mg/kg of INX P linker payload).

INX234P treatment improves mouse survival INX234P treatment was also shown to prevent weight loss compared to hIgG1 and naked Ab control groups, which was interpreted as improved survival. At the end of treatment, INX234P-treated mice began to lose weight. INX234P treatment resulted in improved survival as shown by median survival of 52 days vs. 38.5 and 42 days for the human IgG1 and INX234 treated groups, respectively.

As shown by the experimental results in Figure 120, INX234P treatment improves mouse survival. In these experiments, mice were administered 10 mg/Kg (or 0.2 mg/Kg INX P linker payload) intraperitoneally once a week from day 0 to day 34. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve, with the upper ( The gray bar below (bottom graph) or below (top graph) indicates the treatment period (SEM, n=8/group).

Experiment 2
INX234V/P Treatment Prevents Human PBMC Proliferation In this experiment, animals were treated with INX234V once a week until day 27, then treated with INX234P for the remainder of the experiment, and then (>98%), human T cell proliferation was followed over time. As shown in Figure 121, dramatic differences in cell numbers can be observed as early as day 15. While both control groups showed stagnant cell proliferation at day 28, the INX234V/P treated group showed consistently low T cell numbers.

Specifically, in the experiment of Figure 121, peripheral blood was collected weekly starting from day 15 post-transplant, and human CD45+CD3+ positive cells were quantified by flow cytometry. Mice were dosed once a week from day 0 (SEM, n=8/group) (dose at 10 mg/Kg, INX234V and INX234P received 0.2 mg/kg of INX V or INX P linker payload, respectively). provided).

INX234V/P treatment improves mouse survival INX234V/P treatment also completely prevented weight loss compared to hIgG1 and unconjugated Ab control groups, which translated into a dramatic survival improvement ( Figure 122), the median survival times for human IgG1 and unconjugated INX234 were 42 and 44.5 days, respectively. All animals in the INX234V/P treatment group were alive at the time of this report.

As shown by the results in Figure 122, INX234V/P treatment improves mouse survival. In these experiments, mice were dosed intraperitoneally at 10 mg/Kg (or 0.2 mg/Kg INX P linker payload) once a week starting from day 0. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve.

Conclusions Data show that both INX234P and INX234V can control the progression of heterologous GvHD and increase survival by limiting/preventing human T cell proliferation and disease development in immunodeficient mice. The data further show that changing the linker payload from INX V to INX P in Experiment 2 maintained therapeutic efficacy. This efficacy is mediated by the GC payload, as shown by the fact that naked antibody INX234 elicited no efficacy.

References 1-Johnston RJ, Su LJ, Pinckney J, Critton D, Boyer E, Krishnakumar A, Corbett M, Rankin AL, Dibella R, Campbell L, Martin GH, Lemar H, Cayton T, Huang RY, Deng X, Nayeem A, Chen H, Ergel B, Rizzo JM, Yamniuk AP, Dutta S, Ngo J, Shorts AO, Ramakrishnan R, Kozhich A, Holloway J, Fang H, Wang YK, Yang Z, Thiam K, Rakestraw G, Rajpal A, Sheppard P, Quigley M, Bahjat KS, Korman AJ. “VISTA is an acidic pH-selective ligand for PSGL-1.”Nature. 2019 Oct;574(7779):565-570.
2-McPherson MJ, et al. (2017) Glucocorticoid receptor agonist and immunoconjugates thereof, US15/611,037

Example 25: Anti-VISTA antibody drug conjugate has efficacy in mouse T-cell transplant colitis model Inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis) is a chronic inflammation of the intestine and/or colon. It is a sexual disease. Glucocorticoids (GCs) are highly effective in the treatment of IBD, but these powerful anti-inflammatory drugs are usually limited to the treatment of acute flares due to the toxicity associated with long-term treatment.

Naive T cell transplanted mouse models of chronic colitis have become a means of delineating the immunological mechanisms involved in the induction and regulation of intestinal inflammation. It is also the only mouse model of colitis that is highly responsive to GC.

The efficacy of two antibody drug conjugates (ADCs) INX234P and INX234V, both anti-human VISTA monoclonal antibodies conjugated to two different glucocorticoid (GC) payloads, was evaluated in a T cell transplantation mouse model of colitis. .

Briefly, naive CD4 T cells from human VISTA expressing mice (hVISTA KI) were transplanted into immunodeficient Rag1-/- mice that do not produce mature T cells, B cells, and NK cells. After >21 days, animals begin to slowly lose weight and a decrease in circulating naive T cells is observed. In this model, ADCs target only transplanted cells, and free GCs can act on both transplanted and host cells.

In experiment 1, mice were treated with 10 mg/Kg INX234P once a week starting on day 0. In Experiment 2, treatment with INX234V began on day 21 at the first sign of disease and was treated weekly thereafter. Four control groups were included in both experiments. PBS, 2 and 0.2 mg/Kg dexamethasone (Dex), and unconjugated INX234 antibody were all administered once weekly. In Experiment 1, treatment started on day 0 (prophylactic) and ended on day 61, whereas in Experiment 2, treatment started on day 21 (or therapeutic) and is ongoing at the time of this application. It remains as it is.

The data obtained in both experiments show that both INX234P and INX234V have a superior effect below compared to either dose of Dex.
1) Maintaining the transplanted naïve CD4 T cell population 2) Protecting against disease as shown by dramatically increased survival

Moreover, the data also show that targeting only disease effector cells is sufficient to prevent disease development.

Materials and Methods Study Design In both experiments, all treatments were administered once weekly.

For experiment 1, INX234P treatment was started on day 0 (or prophylactically) after naive CD4 T cell transplantation and ended on day 61. For experiment 2, INX234V treatment was started on day 21 (or therapeutically) after naive CD4 T cell transplantation. In this experiment, on days 42, 49, and 56, animals received INX234P and then returned to INX234V for the remainder of the treatment period.
Test drug and dosage Antibody INX234 (ATUM, lot number 72931.2.a) is a humanized anti-human VISTA antibody on a human IgG1/kappa backbone with L234A/L235A/E269R/K322A silencing mutations in the Fc region. be.
• INX234P (Abzena, lot number JZ-0556-017-2) is INX234, with a drug/antibody ratio (DAR) of 8.0, conjugated through full modification of the interchain disulfide. The linker/payload (INX P) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-3).
• INX234V (Abzena, lot number RJS-1054-003) is an INX234 antibody with a DAR of 7.9 that is conjugated via interchain disulfide modification. The linker/payload (INX V) consists of a negatively charged protease-sensitive linker with a budesonide analog payload (INX-SM-32).

All antibodies were diluted in PBS and injected intraperitoneally (i.p.) in a volume of 0.2 ml to deliver a dose of 10 mg/Kg.

Dexamethasone Dexamethasone sterile injection from Phoenix, NDC 57319-519-05 was diluted in PBS to a final volume of 0.2 ml and administered as described via intraperitoneal injection.
Mice • Eight week old female Rag1-/- mice or B6.129S7-Rag1 ^tm1Mom /J were ordered from Jackson Laboratories (stock number 002216).
- Human VISTA knock-in (hVISTA KI) mice have human VISTA cDNA knocked in instead of the mouse VISTA gene and express human VISTA at both RNA and protein levels with the same expression pattern as mouse VISTA or C57Bl/6 mice. hVISTA KI mice were bred at the Center for Comparative Medicine and Research at Dartmouth. Female mice aged 8-10 weeks were used for cell transplantation.

Disease Model Setup Naive CD4 T Cell Isolation Naive CD4 T cells were isolated from hVISTA KI using the EasySep™ Mouse Naive CD4+ T Cell Isolation Kit from StemCell (Cat. No. 19765) according to the manufacturer's instructions. Isolated from spleen.

Initiation of colitis 0.5 x 106 naive CD45RB+, CD4+ T cells were injected intraperitoneally on day 0 according to the protocol of Ostanin et al (2008).

Whole blood immunostaining Peripheral blood was collected from the retroorbital cavity using a glass Pasteur pipette that was first rinsed with heparin to prevent clotting.

The single wash protocol described below allows for absolute blood cell counts.

10 ml of antibody cocktail (see table on the right) was added directly to 100 ml of blood. After 30 minutes of incubation at room temperature (RT), 600 ml of BD FACS lysis buffer was added to the samples. After incubation for 30 minutes at room temperature, samples were spun at 550 rcf for 5 minutes, washed once in PBS, and then resuspended in a fixed volume of PBS. The entire sample was run on a MacsQuant flow cytometer to obtain absolute cell counts.

Disease Monitoring Mice were weighed three times a week and euthanized when body weight fell below 75% of initial body weight, as allowed by the IACUC organization.

Results Experiment 1
INX234P Treatment Improves Survival As shown in Figures 123 and 124, the control, PBS, and unconjugated (Experiment 1), and low Dex (0.2 mg/Kg) groups, respectively, Median survival times were 74.5 days and 72.5 days. In contrast, by the end of the experiment on day 91, neither the INX234P nor the high Dex (2 mg/Kg) group had reached median survival. Additionally, 20% of the mice (2 out of 10) had to be sacrificed in the high-Dex group, whereas all mice survived in the INX234P-treated group (1 censored due to early unrelated events). (excluding animals). By 10 days after the end of treatment on day 61, INX234P-treated animals began to lose weight (Figures 123 and 124).

More specifically, in the experiment of Figure 123, mice received both INX234P and INX234 at 10 mg/Kg (and 0.2 mg/Kg if applicable) once a week from day 0 to day 61. INX P linker payload) was administered intraperitoneally. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve, with the upper ( The gray bar below (bottom graph) or below (top graph) indicates the treatment period (SEM, excluding INX234P group containing n=9, n=10/group).

The data in Figure 124 show that high dose dexamethasone treatment improved mouse survival. In the experiment, mice were dosed intraperitoneally at 2 (high) or 0.2 (low) mg/Kg once a week from day 0 to day 61. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve, with the upper ( The gray bar below (bottom graph) or below (top graph) indicates the treatment period (SEM, n=10/group).

Experiment 2
This experiment evaluated the ability of anti-VISTA conjugated to linker payload INX V to provide therapeutic efficacy by starting treatment 21 days after naive T cell transplantation. On days 42, 49, and 56, animals received INX234P and were then switched back to INX234V for the remainder of the treatment period.

INX234V Treatment Improves Survival As shown by the data in Figures 125 and 126, the body weight of animals treated with INX234V increased steadily over the course of the experiment (which is normal), whereas all other The group showed some weight gain during the first 30 days followed by a decrease over time.

Although median survival had not been reached in most groups at the time of reporting, the fact that there was no weight loss in the ADC-treated group is likely to be interpreted as a dramatic improvement in survival. Additionally, surprisingly, the group treated with Dex at high doses showed decreased survival when compared to all other groups, including the PBS group (Figure 126).

The data in Figure 125 show that INX234V treatment improves mouse survival. In these experiments, mice were treated with 10 mg/Kg of both INX234V and INX234 (and 0.2 mg/Kg of INX V linker payload, if applicable) once a week from day 21 to day 80. It was administered intraperitoneally. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve, with the upper ( The gray bar below (bottom graph) or below (top graph) indicates the treatment period (SEM, excluding INX234 treatment group where n=5, n=10/group).

The data in Figure 126 show that low dose dexamethasone treatment improves mouse survival. In these experiments, mice were dosed intraperitoneally at 2 (high) or 0.2 (low) mg/Kg once a week from day 21 to day 80. The top left graph shows the average weight change as a percentage of initial body weight, the three bottom graphs show the % weight loss of individual mice, and the top right graph is the Kaplan-Meier survival curve, with the upper ( The gray bar below (bottom graph) or below (top graph) indicates the treatment period (SEM, n=10/group).

INX234V treatment does not affect the survival of transplanted T cells, but limits their activation.
Colitis development is caused by proliferation and activation of transplanted naive CD4 T cells. To monitor disease development, the number and activation status of T cells in peripheral blood were measured on a weekly basis. T cell activation was measured as loss of CD45RB expression, a naive cell marker. Treatment was started on day 21 when we observed a sharp drop in the frequency of CD45 ^RB+ CD4 T cells compared to day 7, when approximately 50% of T cells were still CD45 ^RB+ (Figure 127, bottom graph ). Animals were randomized at enrollment for treatment.

As shown in Figure 127, although there is no difference in T cell numbers between the PBS control and Dex-treated groups (top right graph), INX234V-treated mice exhibit a very consistent and limited proliferation of T cells. (upper left graph). The group treated with unconjugated antibody showed lower T cell numbers only during the first two weeks of treatment (top left graph). In addition, the frequency of CD45 ^RB+ T cells or naïve T cells decreased dramatically between days 15 and 21 in all groups before treatment initiation, and INX234V treatment engrafted the naïve T cell population. (bottom left graph), while all other groups showed a frequency of naïve cells of <10% (bottom left and bottom right graphs). .

The data in Figure 127 show that INX234V treatment prevents T cell proliferation and activation. In these experiments, mice were treated with INX234V or INX234 at 10 mg/Kg (and 0.2 mg/Kg V linker payload if applicable) and 2 (high ) or 0.2 (low) mg/Kg of Dex intraperitoneally. The top left graph shows the number of naïve T cells from blood for INX234V and INX234 vs. PBS on the left and Dex at high and low doses on the right, and the bottom left and right graphs show the number of CD45 ^RB+ CD4 T cells for the same group. The gray bar above indicates the treatment period (SEM, excluding INX234 treatment group where n=5, n=10/group).

Conclusion The data show that both INX234P and INX234V have superior effects compared to Dex, both prophylactically and therapeutically.
1) Maintaining the transplanted naïve CD4 T cell population 2) Protecting against disease as shown by dramatically increased survival

Moreover, the data also show that targeting only disease effector cells is sufficient to prevent disease development.

References - McPherson MJ, et al. (2017) Glucocorticoid receptor agonist and immunoconjugates thereof, US15/611,037
-Dmitry V. Ostanin et al. (2009) T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade, Am J Physiol Gastrointest Liv er Physiol. ;296(2):G135-G146.

Summary of Exemplary Advantages of the Subject ADCs Experimental results disclosed in this application demonstrate that the subject ADCs are ADCs that target anti-inflammatory agents, particularly steroids, to immune cells, such as CD74, CD163, TNF, and PRLR. VISTA as an ADC target, and the anti-VISTA contained in the subject ADCs. This is due to the combined advantages of specific properties of the antibody: it binds to VISTA-expressing immune cells at physiological pH and has a very short pK.

These benefits include:
1) The subject ADCs bind VISTA-expressing immune cells at very high densities and are effective (eliciting anti-inflammatory activity) for long periods of time despite their very short PK (long PD) and are therefore well suited for treating chronic or episodic inflammatory or autoimmune or allergic diseases where long-term and repeated administration is therapeutically warranted.
2) The subject ADCs target a wide range of immune cells, including neutrophils, myeloid, T cells, and endothelium, and therefore the subject ADCs involve any or all of these types of immune cells It can be used to treat inflammatory or autoimmune or allergic diseases.
3) The subject ADCs have a rapid onset of efficacy (as little as 2 hours) and therefore can be used for acute treatment.
4) The subject ADCs do not bind to B cells and therefore should not be immunosuppressive like free steroids (ie, humoral immunity is preserved). This potentially reduces toxicity or adverse side effects during chronic or long-term use of the subject ADC that are associated with long-term use of free steroids (e.g., long-term steroid use is clearly associated with long-term immunosuppression). due to side effects that have been correlated with some cancers, infectious conditions, and other diseases).
5) The subject ADC acts on Tregs, key immune cells involved in steroid efficacy.
6) The subject ADCs include myeloid cells, monocytes, eosinophils, dendritic cells, Tregs, CD8 T cells, CD4 T cells, NK cells, macrophages, neutrophils (in which they are constitutively expressed), etc. As a result, the subject ADCs are active (inducing anti-inflammatory activity) in both active and remission phases of inflammatory and autoimmune conditions.
7) The subject ADCs act on neutrophils, immune cells essential for acute inflammation, and the ADCs are useful for the treatment of acute inflammation, as well as inflammation associated with the active phase of, e.g., chronic or episodic autoimmunity or inflammatory conditions. It further proves that it is well suited to control seizures at an early stage of onset, ideally before pathological symptoms appear. This potentially reduces tissue damage that can occur even before the subject experiences pain or other symptoms related to inflammation.
8) Due to high VISTA cell surface turnover, the subject ADCs internalize immune cells very rapidly and constitutively.
9) The subject ADCs possess a very short half-life (PK) and bind only to immune cells; therefore, the subject ADCs are less susceptible to target-related toxicity and undesirable peripheral steroid exposure (low non-specific loss effects) There should not be a tendency.
10) The subject ADC biological activity (anti-inflammatory effect) of some embodiments is that anti-VISTA antibodies carrying silent IgG therein exhibit no immunological function (blocking of any VISTA biology). anti-inflammatory payloads (steroids), thereby potentially simplifying administration and/or avoiding potentially harmful side effects, e.g. ) is entirely due to.
11) In some embodiments, the biological activity (anti-inflammatory or immunosuppressive effect) of the subject ADCs is attributable to both the anti-inflammatory payload (steroid) and the Fc portion of the anti-VISTA antibody, in particular In one form, the anti-VISTA antibody contains a functional IgG2 Fc region, which indicates that the binding of the anti-VISTA antibody carrying functional IgG2 to VISTA-expressing immune cells promotes the immunosuppressive effects of VISTA, particularly T-cell proliferation and T-cell proliferation. This is to provide an ADC that operates on its suppressive effect on cellular activity, thereby having immunosuppressive activity induced by two different mechanisms.
- Advantages of anti-VISTA ADC 1) VISTA is highly expressed on selected immune cells.
o Targets the drug to neutrophils, bone marrow, T cells, and endothelium o Does not bind to B cells and therefore may not be immunosuppressive like free glucocorticoids.
o Expressed on Tregs (the main cell type target of glucocorticoids in lymphocytes) o One of the only targets expressed on neutrophils (essential for acute inflammation)
○ Internalize rapidly and constitutively.
○VISTA cell surface turnover is also high.
2) Anti-VISTA reduces any target-related toxicity due to its short half-life and reduces peripheral GC exposure (low non-specific loss effect)
3) Silent IgG shows no immunological function (no blockade of any VISTA biology)
Other binding antibody ADC candidates benefit from glucocorticoid linker payloads with independent functions for targets: CD74, CD163, TNF, PRLR INX P series (INX P, INX S)
1) Enhancement of intracellular levels of released payload over literature comparisons. 2) Enhancement of potency of conjugated linker payload over conjugated comparison linker payload, although comparator free payload has similar potency.
3) Allows for a stable drug-antibody ratio of 8 with little or no aggregation at 150 mg/mL.
○ Stable, high-concentration formulations enable the future of subcutaneous administration in the INX V series (INX V, INX A3, INX A11, INX A7, INX A23, INX A12)
1) High enhancement of intracellular levels of released payload over literature comparisons 2) Conjugated linker payloads have enhanced potency over other conjugated linker payloads with payloads of similar potency .
Advantages of glucocorticoid anti-VISTA ADCs 1) Provides long intracellular exposure of glucocorticoids due to high internalization into immune cells 2) Limited GC to non-immune cells due to <24 hour serum half-life of ADCs 3) Improved administration due to higher potency of the glucocorticoid pathway resulting from limited intracellular metabolism of the drug 4) Stable drug-antibody ratio of 8 with little or no aggregation at 150 mg/mL.
○ Stable and highly concentrated formulations enable future potential for subcutaneous administration

References Cited in the Examples The following references and all other references cited in this application are incorporated by reference in their entirety.
(1) Johnston, R. J. et al. W. O. Publication No. 2018/169993A1.
(2) Graversen, J. H. et al. Mol Ther. 2012 Aug;20(8):1550-1558
(3) Vafa, O. et al. Methods. 2014 Jan; 65(1): 114-26.
(4) Durbin, K. R. , Phipps, C. , & Liao, X. (2018). Mechanistic Modeling of Antibody-Drug Conjugate Internalization at the Cellular Level Reveals Inefficient Processing St eps. Mol Cancer Ther, 1535-7163.
(5) Liao-Chan, S. , Daine-Matsuoka, B. , Heald, N. , Wong, T. , Lin, T. , Cai, A. G. 、． ．． ．． Theunissen, J. W. (2015). Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores. PLoS One, 10(4):e012470.
(6) Liu, Z. , Yu, Z. , He, W. Liu, Z. , Yu, Z. , He, W. , Ma, S. , Sun, L. , & Wang, L. (2009). “In-vitro internalization and in-vivo tumor uptake of anti-EGFR monoclonal antibody LA22 in A549 lung cancer cells and ani "mal model". Cancer Biother Radiopharm, 15-23.

配列番号２Ｍｕｓ ｍｕｓｃｕｌｕｓ ＶＩＳＴＡアミノ酸配列

配列番号３Ｍｕｓ ｍｕｓｃｕｌｕｓ ＶＩＳＴＡアミノ酸配列

配列番号４ホモサピエンスＶＩＳＴＡ（別名：Ｂ７－Ｈ５、Ｂ７Ｈ５、ＤＤ１アルファ、ＧＩ２４、ＰＰ２１３５、ＳＩＳＰ１）核酸配列

配列番号５ホモサピエンスＶＩＳＴＡ（別名：Ｂ７－Ｈ５、Ｂ７Ｈ５、ＤＤ１アルファ、ＧＩ２４、ＰＰ２１３５、ＳＩＳＰ１）コード核酸配列

配列番号６Ｍｕｓ ｍｕｓｃｕｌｕｓ ＶＩＳＴＡコード核酸配列

配列番号１０
ＶＳＴＢ９２ ＶＨ
ＱＶＱＬＶＱＳＧＡＥＶＫＫＰＧＡＳＶＫＶＳＣＫＡＳＧＹＴＦＡＮＹＬＩＧＷＶＲＱＡＰＧＱＲＬＥＷＭＧＤＩＹＰＧＧＧＦＩＳＹＮＥＫＦＫＧＲＶＴＩＴＲＤＴＳＡＳＴＡＹＭＥＬＳＳＬＲＳＥＤＴＡＶＹＹＣＡＲＲＦＤＹＧＧＹＦＦＤＹＷＧＱＧＴＬＶＴＶＳＳ
配列番号１１
ＶＳＴＢ９２＿ＶＬ
ＤＩＶＭＴＱＳＰＬＳＬＰＶＴＰＧＥＰＡＳＩＳＣＲＳＳＱＳＩＶＨＳＮＧＮＩＹＬＥＷＹＬＱＫＰＧＱＳＰＱＬＬＩＹＫＶＳＮＲＦＳＧＶＰＤＲＦＳＧＳＧＳＧＴＤＦＴＬＫＩＳＲＶＥＡＥＤＶＧＶＹＹＣＦＱＧＳＨＶＰＷＴＦＧＱＧＴＫＬＥＩＫ
配列番号１２
ＩｇＧ１＿ＬＡＬＡ
ＡＳＴＫＧＰＳＶＦＰＬＡＰＳＳＫＳＴＳＧＧＴＡＡＬＧＣＬＶＫＤＹＦＰＥＰＶＴＶＳＷＮＳＧＡＬＴＳＧＶＨＴＦＰＡＶＬＱＳＳＧＬＹＳＬＳＳＶＶＴＶＰＳＳＳＬＧＴＱＴＹＩＣＮＶＮＨＫＰＳＮＴＫＶＤＫＫＶＥＰＫＳＣＤＫＴＨＴＣＰＰＣＰＡＰＥＬＬＧＧＰＳＶＦＬＦＰＰＫＰＫＤＴＬＭＩＳＲＴＰＥＶＴＣＶＶＶＤＶＳＨＲＤＰＥＶＫＦＮＷＹＶＤＧＶＥＶＨＮＡＫＴＫＰＲＥＥＱＹＮＳＴＹＲＶＶＳＶＬＴＶＬＨＱＤＷＬＮＧＫＥＹＫＣＡＶＳＮＫＡＬＰＡＰＩＥＫＴＩＳＫＡＫＧＱＰＲＥＰＱＶＹＴＬＰＰＳＲＤＥＬＴＫＮＱＶＳＬＴＣＬＶＫＧＦＹＰＳＤＩＡＶＥＷＥＳＮＧＱＰＥＮＮＹＫＴＴＰＰＶＬＤＳＤＧＳＦＦＬＹＳＫＬＴＶＤＫＳＲＷＱＱＧＮＶＦＳＣＳＶＭＨＥＡＬＨＮＨＹＴＱＫＳＬＳＬＳＰＧＫ
配列番号１３
ＩｇＧ１＿ＩＮＸＬＡＬＡ
ＡＳＴＫＧＰＳＶＦＰＬＡＰＳＳＫＳＴＳＧＧＴＡＡＬＧＣＬＶＫＤＹＦＰＥＰＶＴＶＳＷＮＳＧＡＬＴＳＧＶＨＴＦＰＡＶＬＱＳＳＧＬＹＳＬＳＳＶＶＴＶＰＳＳＳＬＧＴＱＴＹＩＣＮＶＮＨＫＰＳＮＴＫＶＤＫＫＶＥＰＫＳＣＤＫＴＨＴＣＰＰＣＰＡＰＥＡＡＧＧＰＳＶＦＬＦＰＰＫＰＫＤＴＬＭＩＳＲＴＰＥＶＴＣＶＶＶＤＶＳＨＲＤＰＥＶＫＦＮＷＹＶＤＧＶＥＶＨＮＡＫＴＫＰＲＥＥＱＹＮＳＴＹＲＶＶＳＶＬＴＶＬＨＱＤＷＬＮＧＫＥＹＫＣＡＶＳＮＫＡＬＰＡＰＩＥＫＴＩＳＫＡＫＧＱＰＲＥＰＱＶＹＴＬＰＰＳＲＤＥＬＴＫＮＱＶＳＬＴＣＬＶＫＧＦＹＰＳＤＩＡＶＥＷＥＳＮＧＱＰＥＮＮＹＫＴＴＰＰＶＬＤＳＤＧＳＦＦＬＹＳＫＬＴＶＤＫＳＲＷＱＱＧＮＶＦＳＣＳＶＭＨＥＡＬＨＮＨＹＴＱＫＳＬＳＬＳＰＧＫ
配列番号１４
ＩｇＧ２＿ｓｉｇｍａ
ＡＳＴＫＧＰＳＶＦＰＬＡＰＣＳＲＳＴＳＥＳＴＡＡＬＧＣＬＶＫＤＹＦＰＥＰＶＴＶＳＷＮＳＧＡＬＴＳＧＶＨＴＦＰＡＶＬＱＳＳＧＬＹＳＬＳＳＶＶＴＶＰＳＳＮＦＧＴＱＴＹＴＣＮＶＤＨＫＰＳＮＴＫＶＤＫＴＶＥＲＫＣＣＶＥＣＰＰＣＰＡＰＰＡＡＡＳＳＶＦＬＦＰＰＫＰＫＤＴＬＭＩＳＲＴＰＥＶＴＣＶＶＶＤＶＳＡＥＤＰＥＶＱＦＮＷＹＶＤＧＶＥＶＨＮＡＫＴＫＰＲＥＥＱＦＮＳＴＦＲＶＶＳＶＬＴＶＬＨＱＤＷＬＮＧＫＥＹＫＣＫＶＳＮＫＧＬＰＳＳＩＥＫＴＩＳＫＴＫＧＱＰＲＥＰＱＶＹＴＬＰＰＳＲＥＥＭＴＫＮＱＶＳＬＴＣＬＶＫＧＦＹＰＳＤＩＡＶＥＷＥＳＮＧＱＰＥＮＮＹＫＴＴＰＰＭＬＤＳＤＧＳＦＦＬＹＳＫＬＴＶＤＫＳＲＷＱＱＧＮＶＦＳＣＳＶＭＨＥＡＬＨＮＨＹＴＱＫＳＬＳＬＳＰＧＫ
配列番号１５
ヒト＿カッパ＿定常
ＲＴＶＡＡＰＳＶＦＩＦＰＰＳＤＥＱＬＫＳＧＴＡＳＶＶＣＬＬＮＮＦＹＰＲＥＡＫＶＱＷＫＶＤＮＡＬＱＳＧＮＳＱＥＳＶＴＥＱＤＳＫＤＳＴＹＳＬＳＳＴＬＴＬＳＫＡＤＹＥＫＨＫＶＹＡＣＥＶＴＨＱＧＬＳＳＰＶＴＫＳＦＮＲＧＥＣ Unofficial sequence list SEQ ID NO: 1 Homo sapiens VISTA (also known as: B7-H5, B7H5, DD1 alpha, GI24, PP2135, SISP1) amino acid sequence

SEQ ID NO: 2 Mus musculus VISTA amino acid sequence

SEQ ID NO:3 Mus musculus VISTA amino acid sequence

SEQ ID NO: 4 Homo sapiens VISTA (also known as: B7-H5, B7H5, DD1 alpha, GI24, PP2135, SISP1) nucleic acid sequence

SEQ ID NO: 5 Homo sapiens VISTA (alias: B7-H5, B7H5, DD1 alpha, GI24, PP2135, SISP1) encoding nucleic acid sequence

SEQ ID NO: 6 Mus musculus VISTA encoding nucleic acid sequence

Sequence number 10
VSTB92 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFANYLIGWVRQAPGQRLEWMGDIYPGGGFISYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRFDYGGYFFDYWGQGTLVTVSS
Sequence number 11
VSTB92_VL
DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNIYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKLEIK
Sequence number 12
IgG1_LALA
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHRDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Sequence number 13
IgG1_INXLALA
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHRDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Sequence number 14
IgG2_sigma
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFP PKPKDTLMISRTPEVTCVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Sequence number 15
Human_Kappa_StationaryRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims (132)

A glucocorticoid agonist compound of formula (I) having the following structure,

During the ceremony,
X is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C ₁ further substituted with _~3 alkyl or C1-3 perfluoroalkyl,
Z is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C ₁ further substituted with _~3 alkyl or C1-3 perfluoroalkyl,
Y is selected from CHR ₁ , O, S, and NR ₁ ,
E is selected from CH ₂ and O;
G is selected from CH and N;
Furthermore, when G is CH and X is phenyl, Z is not phenyl,
The bond _of G to optionally further substituted with 1 to 4 C _1-3 alkyl,
the bond of X to Z may occupy any available position on X and Z;
The substituent NR ₁ R ₂ may occupy any available position on Z;
R ₁ is selected from H, linear or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and said aryl and heteroaryl groups are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, - OH, -O-alkyl, _-NH2 , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C( O)-, and dialkylamino C(O)-,
When R ₁ is H, R ₂ may be selected from H, linear or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and the aryl and heteroaryl groups are alkyl, haloalkyl, Halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH ₂ , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O )O-, alkylamino-C(O)-, and dialkylamino-C(O)-,
When R ₁ is H, linear or branched alkyl of 1 to 8 carbons, or heteroaryl, R ₂ is
[(C=O)CH(W)NH] _m -[C=O]-[V] _k -J,
C=O)OCH ₂ -p-aminophenyl-NVJ,
(C=O)OCH ₂ -p-aminophenyl-N-[(C=O)CH(W)NH] _m -[C=O]-[V] _k -J, and [V] _k -(C =O)OCH ₂ -p-aminophenyl-N-[(C=O)CH(W)NH] _m -[C=O]-J,
may be a functional group selected from;
where m=1 to 6, k=0 to 1, and each permutation of W is independently H, n=1 to 4 [(CH ₂ ) _n R ₃ ], R ₃ branched alkyl chains terminating in and linear or branched polyethylene oxide groups containing from 1 to 13 units;
R ₃ is H, methyl, ethyl, isopropyl, OH, O-alkyl, NH ₂ , NH-alkyl, N-dialkyl, SH, S-alkyl, guanidine, urea, carboxylic acid, carboxamide, carboxylic acid ester, substituted or selected from unsubstituted aryl, substituted or unsubstituted heteroaryl, wherein said aryl and heteroaryl substituents are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, _-NH2 , alkylamino, Selected from dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino C(O)- can be done,
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein said aryl and the heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH ₂ , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, may be substituted with a functional group selected from alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH ₂ , N ₃ , thio, cyclooctyne, -OH, -CO ₂ H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R ₃₂ is selected from Cl, Br, F, mesylate, and tosylate, and R ₃₃ is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-( 4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, R ₃₄ is H, Me, tetrazine-H, and tetrazine-Me;
R ₅ is -CH ₂ OH, -CH ₂ SH, -CH ₂ Cl, -SCH ₂ Cl, -SCH ₂ F, -SCH ₂ CF ₃ , hydroxy, -OCH ₂ CN, -OCH ₂ Cl, -OCH ₂ F, -OCH ₃ , -OCH ₂ CH ₃ , -SCH ₂ CN, and

selected from the group consisting of
R ₆ and R ₇ are independently selected from hydrogen and C _1-10 alkyl;
Q is H,

R ₈ is straight chain or branched alkyl of 1 to 8 carbons, C(O)R ₈ , or n=1 to 4, and R ₄ =H, alkyl or branched alkyl, or P(O)OR ₄ , (C=O)NR ₄ CH _n NR ₄ (C=O)OCH ₂ -(V) _n -J,
A ₁ and A ₂ are independently selected from H and F;
The aforementioned glucocorticoid agonist compounds, in which all possible stereoisomers are claimed, unless otherwise specified. X and Z are independently selected from phenyl, spiro[3.3]heptane, [1.1.1]bicyclopentane, and bicyclo[2.2.2]octane, and Y is _CH2 and O and the permutations of W are independently selected from CH ₂ CH ₂ CO ₂ H and H, and further, when G is CH and X is phenyl, Z is not phenyl. 1. The glucocorticoid agonist compound according to 1. selected from any of the glucocorticoid agonist compounds of claim 1 or 2, or the glucocorticoid agonist compounds disclosed in Example 3, excluding INX J and INX L, or FIG. 11 or FIG. 118A - Selected from those shown in O. A glucocorticoid agonist compound selected from the INX-steroid payload, INX-steroid linker, and INX-antibody drug conjugate (ADC) compounds disclosed herein, excluding INX J and INX L. below:

A glucocorticoid agonist compound selected from: The preceding claim is attached directly or indirectly to at least one cleavable or non-cleavable peptide and/or non-peptide linker (i.e., "steroid-linker payload" or "glucocorticosteroid-linker payload") The glucocorticoid agonist compound according to any one of paragraphs. At least one cleavable or non-cleavable linker (“L”), optionally a “heterobifunctional group” that is a chemical moiety used to connect said linker in a compound to an antibody or antibody fragment or a compound (steroid-linker payload) comprising a "heterotrifunctional group""Q" and at least one anti-inflammatory agent ("AI"), where "AI" has the following structure:
Q-L-AI or AI-L-Q
The compound (steroid-linker payload) which is a glucocorticoid agonist compound according to any one of claims 1 to 6, which may be represented by: 8. The steroid-linker payload of claim 6 or 7, wherein the linker is selected from any of those disclosed herein or shown in the steroid-linker compounds of FIGS. 118A-E. PAB and/or amino acids or peptides, optionally 1 to 12 amino acids, more optionally dipeptides, tripeptides, quatrapeptides, pentapeptides, more optionally Gly, Asn, Asp, Gln, Leu, Lys, Ala, Phe, Cit, Val, Val-Cit, Val-Ala, Val-Gly, Val-Gln, Ala-Val, Cit-Cit, Lys-Val-Cit, Asp-Val-Ala, Ala-Ala-Asn, Asp-Val-Ala, Ala-Val-Cit, Ala-Asn-Val, βAla-Leu-Ala-Leu, Lys-Val-Ala, Val-Leu-Lys, Asp-Val-Cit, at least one cleavable or non-cleavable linker selected from Val-Ala-Val, and Ala-Ala-Asn, or optionally at least one of GlcA, PAB, and Glu-Gly. , the steroid-linker payload of claim 6, 7, or 8. at least one glucocorticoid agonist compound according to any one of the preceding claims, comprising at least one cleavable linker and/or a sacrificial linker attached directly or indirectly to the glucocorticoid agonist steroid compound. Including steroid-linker payload. 118A-O, excluding INX J and INX L, selected from any of the glucocorticoid agonist compounds or steroid-linker payload compounds disclosed in Example 3 or FIGS. 118A-O. glucocorticoid agonist compound or steroid-linker payload. A glucocorticoid agonist (payload) linker conjugate, comprising:
(i) INX-SM-3-GluGly-alkoxyamine, INX-SM-4-GluGly-alkoxyamine, INX-SM-53-GluGly-alkoxyamine, INX-SM-54-GluGly-alkoxyamine, INX-SM -56-GluGly-alkoxyamine, INX-SM-98-GluGly-alkoxyamine, INX-SM-6-GluGly-alkoxyamine, INX-SM-2-GluGly-alkoxyamine, INX-SM-57-GluGly-alkoxy Amine, INX-SM-31-GluGly-alkoxyamine, INX-SM-32-GluGly-alkoxyamine, INX-SM-10-GluGly-alkoxyamine, INX-SM-40-GluGly-alkoxyamine, INX-SM- 34-GluGly-alkoxyamine, INX-SM-28-GluGly-alkoxyamine, INX-SM-27-GluGly-alkoxyamine, INX-SM-35-GluGly-alkoxyamine, INX-SM-8-GluGly-alkoxyamine , INX-SM-7-GluGly-alkoxyamine, INX-SM-33-GluGly-alkoxyamine, or said Glu-Gly is replaced with a different cleavable peptide linker, and/or another INX or INX-SM A glucocorticoid agonist (payload) linker in which the payload is replaced with an INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O or according to Formulas I, II, or III. conjugate, or (ii) INX-SM-53-GluGly-bromoacetyl, INX-SM-3-GluGly-bromoacetyl, INX-SM-54-GluGly-bromoacetyl, INX-SM-1-GluGly-bromoacetyl , INX-SM-4-GluGly-bromoacetyl, INX-SM-2-GluGly-bromoacetyl, INX-SM-47-GluGly-bromoacetyl, INX-SM-7-GluGly-bromoacetyl, INX-SM-8 -GluGly-bromoacetyl, INX-SM-56-GluGly-bromoacetyl, INX-SM-32-GluGly-bromoacetyl, INX-SM-6-GluGly-bromoacetyl, INX-SM-10-GluGly-bromoacetyl, INX-SM-33-GluGly-bromoacetyl, INX-SM-31-GluGly-bromoacetyl, INX-SM-35-GluGly-bromoacetyl, INX-SM-9-GluGly-bromoacetyl, INX-SM-28- GluGly-bromoacetyl, INX-SM-27-GluGly-bromoacetyl, INX-SM-34-GluGly-bromoacetyl, INX-SM-35-GluGly-bromoacetyl, INX-SM-40-GluGly-bromoacetyl, or The Glu-Gly is replaced with a different cleavable peptide linker and/or another INX or INX-SM payload is optionally from that of FIGS. a glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, selected from
(iii) INX-SM-53-GluGly-dibenzocyclooctyne, INX-SM-1-GluGly-dibenzocyclooctyne, INX-SM-4-GluGly-dibenzocyclooctyne, INX-SM-54-GluGly-dibenzocyclooctyne , INX-SM-7-GluGly-dibenzocyclooctyne, INX-SM-8-GluGly-dibenzocyclooctyne, INX-SM-2-GluGly-dibenzocyclooctyne, INX-SM-57-GluGly-dibenzocyclooctyne, INX -SM-40-GluGly-dibenzocyclooctyne, INX-SM-34-GluGly-dibenzocyclooctyne, INX-SM-28-GluGly-dibenzocyclooctyne, INX-SM-27-GluGly-dibenzocyclooctyne, INX-SM -35-GluGly-dibenzocyclooctyne, INX-SM-9-GluGly-dibenzocyclooctyne, INX-SM-10-GluGly-dibenzocyclooctyne, INX-SM-31-GluGly-dibenzocyclooctyne, INX-SM-32 -GluGly-dibenzocyclooctyne, INX-SM-33-GluGly-dibenzocyclooctyne, INX-SM-56-GluGly-dibenzocyclooctyne, INX-SM-6-GluGly-dibenzocyclooctyne, INX-SM-3-GluGly - dibenzocyclooctyne, or said GluGly is replaced with a different cleavable peptide linker, and another INX or INX-SM payload is optionally from that of FIGS. 118A-O or according to formulas I, II, or III or (iv) a glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, selected from -GluGly-NHS ester, INX-SM-32-GluGly-NHS ester, INX-SM-33-GluGly-NHS ester, INX-SM-53-GluGly-NHS ester, INX-SM-7-GluGly-NHS ester, INX-SM-8-GluGly-NHS ester, INX-SM-2-GluGly-NHS ester, INX-SM-56-GluGly-NHS ester, INX-SM-6-GluGly-NHS ester, INX-SM-54- GluGly-NHS ester, INX-SM-4-GluGly-NHS ester, INX-SM-53-GluGly-NHS ester, INX-SM-3-GluGly-NHS ester, INX-SM-9-GluGly-NHS ester, INX -SM-40-GluGly-NHS ester, INX-SM-34-GluGly-NHS ester, INX-SM-28-GluGly-NHS ester, INX-SM-34-GluGly-NHS ester, INX-SM-28-GluGly -NHS ester, INX-SM-27-GluGly-NHS ester, INX-SM-35-GluGly-NHS ester, INX-SM-10-GluGly-NHS ester, or the GluGly is replaced with a different cleavable peptide linker and another INX or INX-SM payload is substituted for the INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O or according to Formulas I, II, or III. glucocorticoid agonist (payload) linker conjugate,
(v) INX-SM-1-GluGly-maleimide, INX-SM-3-GluGly-maleimide, INX-SM-4-GluGly-maleimide, INX-SM-8-GluGly-maleimide, INX-SM-2-GluGly -Maleimide, INX-SM-7-GluGly-maleimide, INX-SM-56-GluGly-maleimide, INX-SM-6-GluGly-maleimide, INX-SM-54-GluGly-maleimide, INX-SM-53-GluGly -Maleimide, INX-SM-33-GluGly-maleimide, INX-SM-35-GluGly-maleimide, INX-SM-40-GluGly-maleimide, INX-SM-34-GluGly-maleimide, INX-SM-28-GluGly -Maleimide, INX-SM-27-GluGly-maleimide, INX-SM-35-GluGly-maleimide, INX-SM-9-GluGly-maleimide, INX-SM-10-GluGly-maleimide, INX-SM-31-GluGly - maleimide, INX-SM-32-GluGly-maleimide, INX-SM-57-GluGly-maleimide, or the GluGly is replaced with a different cleavable peptide linker and another INX or INX-SM payload is shown in FIG. 118A an INX-SM payload substituted glucocorticoid agonist (payload) linker conjugate optionally selected from those of ) INX-SM-3-GluGly-tetrazine, INX-SM-53-GluGly-tetrazine, INX-SM-1-GluGly-tetrazine, INX-SM-54-GluGly-tetrazine, INX-SM-6-GluGly-tetrazine , INX-SM-56-GluGly-tetrazine, INX-SM-4-GluGly-tetrazine, INX-SM-10-GluGly-tetrazine, INX-SM-31-GluGly-tetrazine, INX-SM-32-GluGly-tetrazine , INX-SM-33-GluGly-tetrazine, INX-SM-7-GluGly-tetrazine, INX-SM-8-GluGly-tetrazine, INX-SM-9-GluGly-tetrazine, INX-SM-27-GluGly-tetrazine , INX-SM-35-GluGly-tetrazine, INX-SM-2-GluGly-tetrazine, INX-SM-40-GluGly-tetrazine, INX-SM-34-GluGly-tetrazine, INX-SM-28-GluGly-tetrazine , INX-SM-27-GluGly-tetrazine, or said GluGly is replaced with a different cleavable peptide linker, and another INX or INX-SM payload is of FIG. 118A-O, or of formula I, II, or a glucocorticoid agonist (payload) linker conjugate substituted with the INX-SM payload contained therein, optionally selected from III, or (vii) INX-SM-6-GluGly-amine; INX-SM-54-GluGly-amine, INX-SM-4-GluGly-amine, INX-SM-53-GluGly-amine, INX-SM-2-GluGly-amine, INX-SM-56-GluGly-amine, INX-SM-57-GluGly-amine, INX-SM-35-GluGly-amine, INX-SM-27-GluGly-amine, INX-SM-40-GluGly-amine, INX-SM-34-GluGly-amine, INX-SM-28-GluGly-amine, INX-SM-35-GluGly-amine, INX-SM-9-GluGly-amine, INX-SM-10-GluGly-amine, INX-SM-31-GluGly-amine, INX-SM-32-GluGly-amine, INX-SM-33-GluGly-amine, INX-SM-7-GluGly-amine, INX-SM-8-GluGly-amine, INX-SM-1-GluGly-amine, INX-SM-3-GluGly-amine, or the GluGly is replaced with a different cleavable peptide linker, and another INX or INX-SM payload is of the one in FIGS. 118A-O, or of formula I, II, or III or (viii) a glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, optionally selected from: Amine, INX-SM-1-PAB-GluGly-Alkoxyamine, INX-SM-3-PAB-GluGly-Alkoxyamine, INX-SM-2-PAB-GluGly-Alkoxyamine, INX-SM-56-PAB-GluGly -Alkoxyamine, INX-SM-35-PAB-GluGly-alkoxyamine, INX-SM-25-PAB-GluGly-alkoxyamine, INX-SM-27-PAB-GluGly-alkoxyamine, INX-SM-35-PAB -GluGly-alkoxyamine, INX-SM-9-PAB-GluGly-alkoxyamine, INX-SM-10-PAB-GluGly-alkoxyamine, INX-SM-31-PAB-GluGly-alkoxyamine, INX-SM-32 -PAB-GluGly-alkoxyamine, INX-SM-33-PAB-GluGly-alkoxyamine, INX-SM-57-PAB-GluGly-alkoxyamine, INX-SM-7-PAB-GluGly-alkoxyamine, INX-SM -8-PAB-GluGly-alkoxyamine, INX-SM-6-PAB-GluGly-alkoxyamine, INX-SM-54-PAB-GluGly-alkoxyamine, INX-SM-4-PAB-GluGly-alkoxyamine, INX -SM-40-PAB-GluGly-alkoxyamine, INX-SM-34-PAB-GluGly-alkoxyamine, or said GluGly and/or said PAB is replaced with a different cleavable peptide linker or non-peptide linker, and another is replaced with an INX-SM payload contained therein optionally selected from those of FIGS. 118A-O or according to Formulas I, II, or III. a glucocorticoid agonist (payload) linker conjugate of, or (ix) INX-SM-1-PAB-GluGly-bromoacetyl, INX-SM-3-PAB-GluGly-bromoacetyl, INX-SM-2-PAB-GluGly -Bromoacetyl, INX-SM-7-PAB-GluGly-bromoacetyl, INX-SM-8-PAB-GluGly-bromoacetyl, INX-SM-40-PAB-GluGly-bromoacetyl, INX-SM-56-PAB -GluGly-bromoacetyl, INX-SM-6-PAB-GluGly-bromoacetyl, INX-SM-154PAB-GluGly-bromoacetyl, INX-SM-4-PAB-GluGly-bromoacetyl, INX-SM-33-PAB -GluGly-bromoacetyl, INX-PAB-GluGly-bromoacetyl, INX-SM-32-PAB-GluGly-bromoacetyl, INX-SM-10-PAB-GluGly-bromoacetyl, INX-SM-34-PAB-GluGly -Bromoacetyl, INX-SM-31-PAB-GluGly-bromoacetyl, INX-SM-9-PAB-GluGly-bromoacetyl, INX-SM-28-PAB-GluGly-bromoacetyl, INX-SM-27-PAB - GluGly-bromoacetyl, INX-SM-35-PAB-GluGly-bromoacetyl, INX-SM-53-PAB-GluGly-bromoacetyl, or said GluGly and/or said PAB are linked with different cleavable peptide linkers or non-cleavable peptide linkers. an INX-SM substituted with a peptide linker and another INX or INX-SM payload contained therein optionally selected from those of FIGS. 118A-O or according to Formulas I, II, or III. another glucocorticoid agonist (payload) linker conjugate substituted for the payload;
(x) INX-SM-6-PAB-GluGly-dibenzocyclooctyne, INX-SM-54-PAB-GluGly-dibenzocyclooctyne, INX-SM-4-PAB-GluGly-dibenzocyclooctyne, INX-SM-53 -PAB-GluGly-dibenzocyclooctyne, INX-SM-1-PAB-GluGly-dibenzocyclooctyne, INX-SM-7-PAB-GluGly-dibenzocyclooctyne, INX-SM-8-PAB-GluGly-dibenzocyclooctyne , INX-SM-2-PAB-GluGly-dibenzocyclooctyne, INX-SM-56-PAB-GluGly-dibenzocyclooctyne, INX-SM-57-PAB-GluGly-dibenzocyclooctyne, INX-SM-33-PAB -GluGly-dibenzocyclooctyne, INX-SM-32-PAB-GluGly-dibenzocyclooctyne, INX-SM-31-PAB-GluGly-dibenzocyclooctyne, INX-SM-3-PAB-GluGly-dibenzocyclooctyne, INX -SM-9-PAB-GluGly-dibenzocyclooctyne, INX-SM-27-PAB-GluGly-dibenzocyclooctyne, INX-SM-35-PAB-GluGly-dibenzocyclooctyne, INX-SM-34-PAB-GluGly -dibenzocyclooctyne, INX-SM-28-PAB-GluGly-dibenzocyclooctyne, INX-SM-40-PAB-GluGly-dibenzocyclooctyne, INX-SM-10-PAB-GluGly-dibenzocyclooctyne, or the above GluGly and/or said PAB is replaced with a different cleavable peptide linker or non-peptide linker, and another INX or INX-SM payload is any from those in FIGS. 118A-O or according to Formulas I, II, or III. another glucocorticoid agonist (payload) linker conjugate selectively substituted for the INX-SM payload contained therein, or (xi) INX-SM-56-PAB-GluGly-NHS ester; INX-SM-54-PAB-GluGly-NHS ester, INX-SM-4-PAB-GluGly-NHS ester, INX-SM-53-PAB-GluGly-NHS ester, INX-SM-1-PAB-GluGly-NHS ester, INX-SM-3-PAB-GluGly-NHS ester, INX-SM-33-PAB-GluGly-NHS ester, INX-SM-57-PAB-GluGly-NHS ester, INX-SM-7-PAB-GluGly -NHS ester, INX-SM-8-PAB-GluGly-NHS ester, INX-SM-27-PAB-GluGly-NHS ester, INX-SM-35-PAB-GluGly-NHS ester, INX-SM-9-PAB -GluGly-NHS ester, INX-SM-10-PAB-GluGly-NHS ester, INX-SM-31-PAB-GluGly-NHS ester, INX-SM-32-PAB-GluGly-NHS ester, INX-SM-40 -PAB-GluGly-NHS ester, INX-SM-34-PAB-GluGly-NHS ester, INX-SM-28-PAB-GluGly-NHS ester, INX-SM-2-PAB-GluGly-NHS ester, or the above GluGly and/or said PAB is replaced with a different cleavable peptide linker or non-peptide linker, and another INX or INX-SM payload is any from those in FIGS. 118A-O or according to Formulas I, II, or III. another glucocorticoid agonist (payload) linker conjugate selectively substituted for the INX-SM payload contained therein, or (xii) INX-SM-1-PAB-GluGly-maleimide, INX -SM-53-PAB-GluGly-maleimide, INX-SM-5-PAB-GluGly-maleimide, INX-SM-2-PAB-GluGly-maleimide, INX-SM-8-PAB-GluGly-maleimide, INX-SM -56-PAB-GluGly-maleimide, INX-SM-54-PAB-GluGly-maleimide, INX-SM-4-PAB-GluGly-maleimide, INX-SM-57-PAB-GluGly-maleimide, INX-SM-7 -PAB-GluGly-maleimide, INX-SM-32-PAB-GluGly-maleimide, INX-SM-31-PAB-GluGly-maleimide, INX-SM-53-PAB-GluGly-maleimide, INX-SM-3-PAB -GluGly-maleimide, INX-SM-34-PAB-GluGly-maleimide, INX-SM-28-PAB-GluGly-maleimide, INX-SM-40-PAB-GluGly-maleimide, INX-SM-27-PAB-GluGly - maleimide, INX-SM-35-PAB-GluGly-maleimide, INX-SM-9-PAB-GluGly-maleimide, INX-SM-10-PAB-GluGly-maleimide, or the GluGly and/or the PAB are different that is substituted with a cleavable peptide linker or a non-peptide linker, and another INX or INX-SM payload is optionally selected from those of FIGS. 118A-O or according to Formulas I, II, or III. or (xiii) INX-SM-6-PAB-GluGly-Tetrazine, INX-SM-54-PAB-GluGly -tetrazine, INX-SM-4-PAB-GluGly-tetrazine, INX-SM-53-PAB-GluGly-tetrazine, INX-SM-1-PAB-GluGly-tetrazine, INX-SM-3-PAB-GluGly-tetrazine , INX-SM-57-PAB-GluGly-tetrazine, INX-SM-7-PAB-GluGly-tetrazine, INX-SM-8-PAB-GluGly-tetrazine, INX-SM-2-PAB-GluGly-tetrazine, INX -SM-31-PAB-GluGly-tetrazine, INX-SM-32-PAB-GluGly-tetrazine, INX-SM-33-PAB-GluGly-tetrazine, INX-SM-56-PAB-GluGly-tetrazine, INX-SM -35-PAB-GluGly-tetrazine, INX-SM-9-PAB-GluGly-tetrazine, INX-SM-40-PAB-GluGly-tetrazine, INX-SM-34-PAB-GluGly-tetrazine, INX-SM-28 -PAB-GluGly-tetrazine, INX-SM-27-PAB-GluGly-tetrazine, INX-SM-35-PAB-GluGly-tetrazine, INX-SM-10-PAB-GluGly-tetrazine, or said GluGly and/or said PAB is replaced with a different cleavable peptide linker or non-peptide linker, and another INX or INX-SM payload is optionally selected from that of FIGS. 118A-O or according to Formulas I, II, or III another glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, or (xiv) INX-SM-1-PAB-GluGly-amine, INX-SM-3 -PAB-GluGly-amine, INX-SM-8-PAB-GluGly-amine, INX-SM-2-PAB-GluGly-amine, INX-SM-56-PAB-GluGly-amine, INX-SM-6-PAB -GluGly-, or an amine according to formula I, II, or III, INX-SM-54-PAB-GluGly-amine, INX-SM-4-PAB-GluGly-amine, INX-SM-53-PAB-GluGly-amine , INX-SM-33-PAB-GluGly-amine, INX-SM-53-PAB-GluGly-amine, INX-SM-7-PAB-GluGly-amine, INX-SM-9-PAB-GluGly-amine, INX -SM-35-PAB-GluGly-amine, INX-SM-40-PAB-GluGly-amine, INX-SM-34-PAB-GluGly-amine, INX-SM-28-PAB-GluGly-amine, INX-SM -27-PAB-GluGly-amine, INX-SM-35-PAB-GluGly-amine, INX-SM-10-PAB-GluGly-amine, INX-SM-31-PAB-GluGly-amine, INX-SM-32 -PAB-GluGly-amine, or said GluGly and/or said PAB is replaced with a different cleavable peptide linker or a non-peptide linker, and another INX or INX-SM payload is of the formula another glucocorticoid agonist (payload) linker conjugate, optionally selected from those according to I, II, or III, substituted for the INX-SM payload contained therein, or (xv) INX-SM -1-PAB-GlcA-alkoxyamine, INX-SM-35-PAB-GlcA-alkoxyamine, INX-SM-9-PAB-GlcA-alkoxyamine, INX-SM-10-PAB-GlcA-alkoxyamine, INX -SM-54-PAB-GlcA-alkoxyamine, INX-SM-31-PAB-GlcA-alkoxyamine, INX-SM-32-PAB-GlcA-alkoxyamine, INX-SM-33-PAB-GlcA-alkoxyamine , INX-SM-57-PAB-GlcA-alkoxyamine, INX-SM-7-PAB-GlcA-alkoxyamine, INX-SM-8-PAB-GlcA-alkoxyamine, INX-SM-2-PAB-GlcA- Alkoxyamine, INX-SM-56-PAB-GlcA-Alkoxyamine, INX-SM-6-PAB-GlcA-Alkoxyamine, INX-SM-4-PAB-GlcA-Alkoxyamine, INX-SM-53-PAB- GlcA-alkoxyamine, INX-SM-27-PAB-GlcA-alkoxyamine, INX-SM-40-PAB-GlcA-alkoxyamine, INX-SM-34-PAB-GlcA-alkoxyamine, INX-SM-28- PAB-GlcA-alkoxyamine, INX-SM-3-PAB-GlcA-alkoxyamine, or said GlcA and/or said PAB is replaced with a different cleavable peptide linker or non-peptide linker, and/or another INX or the INX-SM payload is replaced with an INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O, or according to Formulas I, II, or III. Corticoid agonist (payload) linker conjugate, or (xvi) INX-SM-3-PAB-GlcA-bromoacetyl, INX-SM-4-PAB-GlcA-bromoacetyl, INX-SM-56-PAB-GlcA-bromo Acetyl, INX-SM-54-PAB-GlcA-bromoacetyl, INX-SM-4-PAB-GlcA-bromoacetyl, INX-SM-53-PAB-GlcA-bromoacetyl, INX-SM-7-PAB-GlcA -Bromoacetyl, INX-SM-8-PAB-GlcA-bromoacetyl, INX-SM-2-PAB-GlcA-bromoacetyl, INX-SM-40-PAB-GlcA-bromoacetyl, INX-SM-57-PAB -GlcA-bromoacetyl, INX-SM-33-PAB-GlcA-bromoacetyl, INX-SM-10-PAB-GlcA-bromoacetyl, INX-SM-34-PAB-GlcA-bromoacetyl, INX-SM-31 -PAB-GlcA-bromoacetyl, INX-SM-32-PAB-GlcA-bromoacetyl, INX-SM-35-PAB-GlcA-bromoacetyl, INX-SM-9-PAB-GlcA-bromoacetyl, INX-SM -28-PAB-GlcA-bromoacetyl, INX-SM-27-PAB-GlcA-bromoacetyl, INX-SM-1-PAB-GlcA-bromoacetyl, or the GluGly and/or the PAB are different cleavable 118A-O or in which the payload is optionally selected from those of FIGS. 118A-O or according to Formulas I, II, or III. or (xvii) INX-SM-4-PAB-GlcA-dibenzocyclooctyne, INX-SM-54-PAB -GlcA-dibenzocyclooctyne, INX-SM-1-PAB-GlcA-dibenzocyclooctyne, INX-SM-54-PAB-GlcA-dibenzocyclooctyne, INX-SM-33-PAB-GlcA-dibenzocyclooctyne, INX -SM-57-PAB-GlcA-dibenzocyclooctyne, INX-SM-7-PAB-GlcA-dibenzocyclooctyne, INX-SM-8-PAB-GlcA-dibenzocyclooctyne, INX-SM-2-PAB-GlcA -dibenzocyclooctyne, INX-SM-5-PAB-GlcA-dibenzocyclooctyne, INX-SM-6-PAB-GlcA-dibenzocyclooctyne, INX-SM-35-PAB-GlcA-dibenzocyclooctyne, INX-SM -9-PAB-GlcA-dibenzocyclooctyne, INX-SM-10-PAB-GlcA-dibenzocyclooctyne, INX-SM-31-PAB-GlcA-dibenzocyclooctyne, INX-SM-32-PAB-GlcA-dibenzo Cyclooctyne, INX-SM-27-PAB-GlcA-dibenzocyclooctyne, INX-SM-35-PAB-GlcA-dibenzocyclooctyne, INX-SM-28-PAB-GlcA-dibenzocyclooctyne, INX-SM-34 -PAB-GlcA-dibenzocyclooctyne, INX-SM-40-PAB-GlcA-dibenzocyclooctyne, INX-SM-3-PAB-GlcA-dibenzocyclooctyne, or said GlcA and/or said PAB can be cleaved differently and/or another INX or INX-SM payload is optionally selected from those of FIGS. 118A-O or according to Formulas I, II, or III. another glucocorticoid agonist (payload) linker conjugate substituted for the INX-SM payload contained therein, or (xviii) INX-SM-3-PAB-GlcA-NHS ester, INX-SM-53-PAB -GlcA-NHS ester, INX-SM-4-PAB-GlcA-NHS ester, INX-SM-56-PAB-GlcA-NHS ester, INX-SM-54-PAB-GlcA-NHS ester, INX-SM-8 -PAB-GlcA-NHS ester, INX-SM-2-PAB-GlcA-NHS ester, INX-SM-7-PAB-GlcA-NHS ester, INX-SM-57-PAB-GlcA-NHS ester, INX-SM -32-PAB-GlcA-NHS ester, INX-SM-33-PAB-GlcA-NHS ester, INX-SM-31-PAB-GlcA-NHS ester, INX-SM-9-PAB-GlcA-NHS ester, INX -SM-10-PAB-GlcA-NHS ester, INX-SM-35-PAB-GlcA-NHS ester, INX-SM-27-PAB-GlcA-NHS ester, INX-SM-28-PAB-GlcA-NHS ester , INX-SM-40-PAB-GlcA-NHS ester, INX-SM-34-PAB-GlcA-NHS ester, INX-SM-1-PAB-GlcA-NHS ester, or the GlcA and/or the PAB, Substituted with a different cleavable peptide linker or non-peptide linker and/or another INX or INX-SM payload optionally selected from those of FIGS. 118A-O or according to Formulas I, II, or III or (xix) INX-SM-3-PAB-GlcA-Maleimide, INX-SM-4 -PAB-GlcA-maleimide, INX-SM-53-PAB-GlcA-maleimide, INX-SM-31-PAB-GlcA-maleimide, INX-SM-32-PAB-GlcA-maleimide, INX-SM-33-PAB -GlcA-maleimide, INX-SM-53-PAB-GlcA-maleimide, INX-SM-7-PAB-GlcA-maleimide, INX-SM-8-PAB-GlcA-maleimide, INX-SM-2-PAB-GlcA -Maleimide, INX-SM-56-PAB-GlcA-maleimide, INX-SM-6-PAB-GlcA-maleimide, INX-SM-54-PAB-GlcA-maleimide, INX-SM-1-PAB-GlcA-maleimide , INX-SM-9-PAB-GlcA-maleimide, INX-SM-35-PAB-GlcA-maleimide, INX-SM-27-PAB-GlcA-maleimide, INX-SM-28-PAB-GlcA-maleimide, INX - SM-34-PAB-GlcA-maleimide, INX-SM-40-PAB-GlcA-maleimide, INX-SM-10-PAB-GlcA-maleimide, or said GlcA and/or said PAB are different cleavable peptides a linker or a non-peptide linker, and/or another INX or INX-SM payload is optionally selected from those of FIGS. 118A-O or according to Formulas I, II, or III. or (xx) INX-SM-33-PAB-GlcA-tetrazine, INX-SM-57-PAB-GlcA- Tetrazine, INX-SM-7-PAB-GlcA-tetrazine, INX-SM-8-PAB-GlcA-tetrazine, INX-SM-2-PAB-GlcA-tetrazine, INX-SM-56-PAB-GlcA-tetrazine, INX-SM-6-PAB-GlcA-tetrazine, INX-SM-54-PAB-GlcA-tetrazine, INX-SM-4-PAB-GlcA-tetrazine, INX-SM-9-PAB-GlcA-tetrazine, INX- SM-35-PAB-GlcA-tetrazine, INX-SM-27-PAB-GlcA-tetrazine, INX-SM-28-PAB-GlcA-tetrazine, INX-SM-34-PAB-GlcA-tetrazine, INX-SM- 40-PAB-GlcA-tetrazine, INX-SM-10-PAB-GlcA-tetrazine, or said GlcAy and/or said PAB are replaced with a different cleavable peptide or non-peptide linker, and/or another INX or the INX-SM payload is replaced with an INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O, or according to Formulas I, II, or III. Corticoid agonist (payload) linker conjugate, or (xxi) INX-SM-1-PAB-GlcA-amine, INX-SM-3-PAB-GlcA-amine, INX-SM-53-PAB-GlcA-amine, INX -SM-6-PAB-GlcA-amine, INX-SM-54-PAB-GlcA-amine, INX-SM-8-PAB-GlcA-amine, INX-SM-2-PAB-GlcA-amine, INX-SM -56-PAB-GlcA-amine, INX-SM-4-PAB-GlcA-amine, INX-SM-35-PAB-GlcA-amine, INX-SM-8-PAB-GlcA-amine, INX-SM-10 -PAB-GlcA-amine, INX-SM-31-PAB-GlcA-amine, INX-SM-32-PAB-GlcA-amine, INX-SM-33-PAB-GlcA-amine, INX-SM-57-PAB -GlcA-amine, INX-SM-27-PAB-GlcA-amine, INX-SM-35-PAB-GlcA-amine, INX-SM-34-PAB-GlcA-amine, INX-SM-28-PAB-GlcA - amine, INX-SM-40-PAB-GlcA-amine, INX-SM-7-PAB-GlcA-amine, or said GlcA and/or said PAB is replaced with a different cleavable peptide or non-peptide linker. , and/or another INX or INX-SM payload contained therein, optionally selected from those of FIGS. 118A-O or according to Formulas I, II, or III. substituted with another glucocorticoid agonist (payload) linker conjugate, or (xxii) alkoxyamine-GlcA-PAB-DMEDA-INX-SM3, or alkoxyamine-GlyGlu-PAB-DMEDA-INX-SM3, or INX- SM3 is substituted with a payload selected from those in FIGS. other INX linker payloads, attached to the same or different INX steroids via
(xxiii) a payload in which bromoacetyl-GlcA-PAB-DMEDA-INX-SM3, or bromoacetyl--GlyGlu-PAB-DMEDA-INX-SM3, or INX-SM3 is selected from those of FIGS. 118A-O; or substituted with another compound of formula I, II, or III and/or containing the same or different peptide or non-peptide linker, said linker being other INX linker payloads attached to the same or different INX steroids,
(xxiv) a payload in which dibenzocyclooctyne-GlcA-PAB-DMEDA-INX-SM3, or dibenzocyclooctyne-GlyGlu-PAB-DMEDA-INX-SM3, or INX-SM3 is selected from those of FIGS. 118A-O; or substituted with another compound of formula I, II, or III and/or containing the same or different peptide or non-peptide linker, said linker being attached via C11-OH to the same or different INX steroid , other INX linker payloads,
(xxv) a payload in which Tetrazine-GlcA-PAB-DMEDA-INX-SM3, or Tetrazine-GlyGlu-PAB-DMEDA-INX-SM3, or INX-SM3 is selected from those of FIGS. 118A-O, or Formula I; Other INXs substituted with another compound of II, or III and/or containing the same or different peptide or non-peptide linker, said linker being attached via C11-OH to the same or different INX steroid linker payload,
(xxvi) a payload or formula in which alkoxyamine-GlcA-PAB-DMEDA-INX-SM3, or alkoxyamine-GlyGlu-PAB-DMEDA-INX-SM3, or INX-SM3 is selected from those of FIGS. 118A-O; other INX linker payloads substituted with another compound of I, II, or III and/or comprising the same or different linker, the linker being attached via C17 to the same or different INX steroid payload;
(xxvii) a payload or formula in which Bromoacetyl-GlcA-PAB-DMEDA-INX-SM3, or Bromoacetyl-GlyGlu-PAB-DMEDA-INX-SM3, or INX-SM3 is selected from those of FIGS. 118A-O; other INX linker payloads substituted with another compound of I, II, or III and/or containing the same or different INX steroid payload via C17;
(xxviii) a payload in which maleimide-GlcA-PAB-DMEDA-INX-SM3, or maleimide-GlyGlu-PAB-DMEDA-INX-SM3, or INX-SM3 is selected from those of FIGS. 118A-O, or Formula I; other INX linker payloads substituted with another compound of II, or III and/or comprising the same or different linker, said linker being attached via C17 to the same or different INX steroid payload;
(xxix) a payload in which dibenzocyclooctyne-GlcA-PAB-DMEDA-INX-SM3, or dibenzocyclooctyne-GlyGlu-PAB-DMEDA-INX-SM3, or INX-SM3 is selected from those of FIGS. 118A-O; or other INX linkers substituted with another compound of formula I, II, or III and/or comprising the same or different linker, said linker being attached via C17 to the same or different INX steroid payload. payload,
(xxx) a payload in which Tetrazine-GlcA-PAB-DMEDA-INX-SM3, or Tetrazine-GlyGlu-PAB-DMEDA-INX-SM3, or INX-SM3 is selected from those of FIGS. 118A-O, or Formula I; other INX linker payloads substituted with another compound of II, or III and/or containing the same or different INX steroid payload via C17;
(xxxi) A payload in which amine-GlcA-PAB-DMEDA-INX-SM3, or amine-GlyGlu-PAB-DMEDA-INX-SM3, or INX-SM3 is selected from those of FIGS. 118A-O, or Formula I; Said glucocorticoid agonist (payload) linker conjugate selected from other INX linker payloads substituted with another compound of II, or III and containing the same or different INX steroid payload via C17. An antibody drug conjugate (ADC) comprising:
(i) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (alkoxyamine + ketone conjugation (C11-OH bond), or INX-SM3 is 118A-O, or another compound of Formula I, II, or III, the other INX-SM payload is conjugated to the antibody via alkoxyamine+ketone conjugation. , another ADC containing a different INX-SM payload, coupled to C11-OH,
(ii) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (azido + dibenzocyclooctyne conjugation (C11-OH bond), or INX-SM3 , or another compound of Formula I, II, or III, and the INX-SM payload is conjugated to the antibody via an azide+dibenzocyclooctyne conjugation. , another ADC containing a different INX-SM payload, coupled to C11-OH,
(iii) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (haloacetyl + cysteine conjugation (C11-OH bond), or INX-SM3 is 118A-O, or another compound of formula I, II, or III, and is conjugated to the antibody via an azide+dibenzocyclooctyne conjugation and is linked to a C11-OH bond. , another ADC containing a different INX-SM payload,
(iv) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (maleimide + cysteine conjugation (C11-OH bond), or INX-SM3 is 118A-O, or another compound of formula I, II, or III, and is conjugated to the antibody via an azide+dibenzocyclooctyne conjugation and is linked to a C11-OH bond. , another ADC containing a different INX-SM payload,
(v) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (tetrazine + trans cyclooctene conjugation (C11-OH bond), or INX-SM3 , a payload selected from those in FIGS. 118A-O, or another compound of formula I, II, or III, and conjugated to the antibody via tetrazine + trans cyclooctene conjugation and C11-OH linked. another ADC containing a different INX-SM payload,
(vi) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (alkoxyamine + ketone conjugation (C11-OH bond)), or INX-SM3 , a payload selected from those in FIGS. another ADC containing a different INX-SM payload,
(vii) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (azido + dibenzocyclooctyne conjugation (C17 bond)), or INX-SM3, substituted with a payload selected from those of FIGS. 118A-O, or another compound of Formula I, II, or III, conjugated to the antibody via an azide+dibenzocyclooctyne conjugation, and C17-linked; another ADC containing a different INX-SM payload,
(viii) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (haloacetyl + cysteine conjugation (C17 bond)), or INX-SM3 is shown in FIG. 118A A different INX substituted with a payload selected from ~O, or another compound of formula I, II, or III and conjugated to the antibody via an azide + dibenzocyclooctyne conjugation and C17-linked. - another ADC containing the SM payload,
(ix) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (tetrazine + trans cyclooctene conjugation (C17 bond)), or INX-SM linker payload Another ADC containing a different INX-SM payload, which is conjugated to the antibody via tetrazine + trans cyclooctene conjugation and C17-linked,
(x) Ab-Gly-Glu-PAB-DMEDA-INX-3 or Ab-GlcA-PAB-DMEDA-INX-SM-3 (amine+gutamine conjugation (C17 linkage) using transglutaminase), or INX - another ADC containing a different INX-SM payload, where the SM linker payload is conjugated to the antibody via amine+glutamine conjugation using transglutaminase and C17-linked;
(xi) INX-SM-3-PAB-GlcA-Ab or INX-SM-3-PAB-Glu-Gly-Ab (alkoxyamine and ketone conjugation) (N-linked payload), or INX-SM3 is shown in FIG. A different INX substituted with a payload selected from those of ~0 or another compound of formula I, II, or III payload and conjugated and N-linked to the antibody via alkoxyamine and ketone conjugation. - another ADC containing the SM payload,
(xii) INX-SM-3-PAB-GlcA-Ab or INX-SM-3-PAB-Glu-Gly-Ab (haloacetyl conjugation) (N-linked payload), or INX-SM3 is or a different INX-SM payload that is substituted with another compound of formula I, II, or III payload and conjugated and N-linked to the antibody via haloacetyl conjugation. another ADC, including
(xiii) INX-SM-3-PAB-GlcA-Ab or INX-SM-3-PAB-Glu-Gly-Ab (azide + dibenzocyclooctyne conjugation) (N-linked payload), or INX-SM3 is 118A-O, or another compound of formula I, II, or III payload and is conjugated and N-linked to the antibody via an azide + dibenzocyclooctyne conjugation, another ADC containing a different INX-SM payload,
(xiv) INX-SM-3-GlcA-Ab or INX-SM-3-Glu-Gly-Ab (N-hydroxysuccinimide conjugation) (N-linked payload), or INX-SM3 as in Figures 118A-O or another compound of formula I, II, or III payload and is conjugated and N-linked to the antibody via N-hydroxysuccinimide conjugation. another ADC, including
(xv) INX-SM-3-PAB-GlcA-Ab or INX-SM-3-PAB-Glu-Gly-Ab (azide + dibenzocyclooctyne conjugation) (N-linked payload), or INX-SM3 is 118A-O, or another compound of formula I, II, or III payload and is conjugated and N-linked to the antibody via an azide + dibenzocyclooctyne conjugation, another ADC containing a different INX-SM payload,
(xvi) INX-SM-3-PAB-GlcA-Ab or INX-SM-3-PAB-Glu-Gly-Ab (N-hydroxysuccinimide conjugation) (N-linked payload), or INX-SM3 is shown in FIG. A different INX substituted with a payload selected from ~O, or another compound of formula I, II, or III payload and conjugated and N-linked to the antibody via N-hydroxysuccinimide conjugation. - another ADC containing the SM payload,
(xvii) INX-SM-3-Glu-Gly-Ab or INX-SM-3-PAB-Glu-Gly-Ab (maleimide conjugation) (N-linked payload), or INX-SM3 as shown in FIGS. or another comprising a different INX-SM payload substituted with another compound of formula I, II, or III payload and conjugated to the antibody via maleimide conjugation and N-linked. ADC,
(xviii) INX-SM-3-Glu-Gly-Ab or INX-SM-3-PAB-Glu-Gly-Ab or INX-SM-3-PAB-GlcA-Ab (trans cyclooctene + tetrazine conjugation) (N-linked payload), or INX-SM3 is substituted with a payload selected from those of FIGS. another ADC containing a different INX-SM payload, conjugated to an antibody via
(xix) INX-SM-3-Glu-Gly-Ab or INX-SM-3-PAB-Glu-Gly-Ab or INX-SM-3-PAB-GlcA-Ab (amine conjugation) (N-linked payload) , or INX-SM3 is substituted with a payload selected from those in Figures 118A-O, or another compound of formula I, II, or III payload and conjugated to the antibody via transcyclooctene+tetrazine conjugation. The antibody drug conjugate (ADC) is selected from another ADC comprising a different INX-SM payload, which is gated and N-linked. An antibody drug conjugate (ADC) selected from:

During the ceremony,
Ab = antibody, preferably an antibody that binds to human immune cells, preferably an anti-VISTA antibody that binds to human VISTA immune cells at physiological pH;
L = linker,
AA = single, double, or triple amino acid sequence;
NH payload =

R ^EG is independently hydrogen, alkyl, biphenyl, -CF ₃ , -NO ₂ , -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino -C(O)-, and dialkylamino C(O)-,

Ab=antibody,
L = linker,
AA = single, double, or triple amino acid sequence;
NH payload=

R ^EG is independently hydrogen, alkyl, biphenyl, -CF ₃ , -NO ₂ , -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino -C(O)-, and dialkylamino C(O)-,

Ab=antibody,
L = linker,
AA = single, double or triple amino acid sequence or absent;
NH payload=

R ^EG is independently hydrogen, alkyl, biphenyl, -CF ₃ , -NO ₂ , -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino -C(O)-, and dialkylamino C(O)-,
RT=AA or

Ab = antibody, optionally anti-human VISTA antibody,
L = linker,
AA = single, double, or triple amino acid sequence;
O payload=

Ab=antibody,
L = linker,
AA = single, double or triple amino acid sequence or absent;
O payload =

RT=AA or

The antibody drug conjugate (ADC). 15. The antibody drug conjugate (ADC) of claim 14, wherein the linker comprises a cleavable or non-cleavable peptide or a sacrificial linker. PAB and/or amino acids or peptides, optionally 1 to 12 amino acids, more optionally dipeptides, tripeptides, quatrapeptides, pentapeptides, more optionally Gly, Asn, Asp, Gln, Leu, Lys, Ala, Phe, Cit, Val, Val-Cit, Val-Ala, Val-Gly, Val-Gln, Ala-Val, Cit-Cit, Lys-Val-Cit, Asp-Val-Ala, Ala-Ala-Asn, Asp-Val-Ala, Ala-Val-Cit, Ala-Asn-Val, βAla-LeuAla-Leu, Lys-Val-Ala, Val-Leu-Lys, Asp-Val-Cit, Val- An antibody drug conjugate (ADC) according to any one of the preceding claims, comprising a linker selected from Ala-Val, and Ala-Ala-Asn. A steroid antibody conjugate selected from the following structures,

In the formula, n=2-12, 2-10, 2-8, 2-6, 2-4, and A is an antibody or an antigen-binding fragment thereof, preferably an immune cell, preferably a human immune cell. The steroid antibody conjugate is an antibody or antibody fragment that binds to an antigen expressed in a human VISTA, more preferably an anti-human VISTA antibody. A glucocorticoid agonist compound of formula (I),

During the ceremony,
X is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C ₁ further substituted with _~3 alkyl or C1-3 perfluoroalkyl,
Z is phenyl, spiro[3.3]heptane, 3- to 6-membered heterocycle, cycloalkyl, spiroalkyl, spiroheterocycloalkyl, bicyclic alkyl, heterobicyclic alkyl, [1.1.1]bicyclo selected from pentane, bicyclo[2.2.2]octane, adamantane, and cuban, each independently selected from 1-4 selected from F, Cl, Br, I, N, S, and O heteroatoms, each of whose ring structures can include at least one backbone heteroatom selected from N, S, and O, optionally with 1 to 4 C ₁ further substituted with _~3 alkyl or C1-3 perfluoroalkyl,
Y is selected from CHR ₁ , O, S, and NR ₁ ,
E is selected from CH ₂ and O;
G is selected from CH and N;
Furthermore, when G is CH and X is phenyl, Z is not phenyl,
The bond _of G to optionally further substituted with 1 to 4 C _1-3 alkyl,
the bond of X to Z may occupy any available position on X and Z;
The substituent NR ₁ R ₂ may occupy any available position on Z;
R ₁ is selected from H, linear or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and said aryl and heteroaryl groups are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, - OH, -O-alkyl, _-NH2 , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O)O-, alkylamino-C( O)-, and dialkylamino C(O)-,
When R ₁ is H, R ₂ may be selected from H, linear or branched alkyl, aryl, and heteroaryl groups of 1 to 8 carbons, and the aryl and heteroaryl groups are alkyl, haloalkyl, Halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, -NH ₂ , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, alkyl-C(O )O-, alkylamino-C(O)-, and dialkylamino-C(O)-,
When R ₁ is H, linear or branched alkyl of 1 to 8 carbons, or heteroaryl, R ₂ is
[(C=O)CH(W)NH] _m -[C=O]-[V] _k -J,
C=O)OCH ₂ -p-aminophenyl-NVJ,
(C=O)OCH ₂ -p-aminophenyl-N-[(C=O)CH(W)NH] _m -[C=O]-[V] _k -J, and [V] _k -(C =O)OCH ₂ -p-aminophenyl-N-[(C=O)CH(W)NH] _m -[C=O]-J,
may be a functional group selected from;
where m=1 to 6, k=0 to 1, and each permutation of W is independently H, n=1 to 4 [(CH ₂ ) _n R ₃ ], R ₃ branched alkyl chains terminating in and linear or branched polyethylene oxide groups containing from 1 to 13 units;
R ₃ is H, methyl, ethyl, isopropyl, OH, O-alkyl, NH ₂ , NH-alkyl, N-dialkyl, SH, S-alkyl, guanidine, urea, carboxylic acid, carboxamide, carboxylic acid ester, substituted or selected from unsubstituted aryl, substituted or unsubstituted heteroaryl, wherein said aryl and heteroaryl substituents are alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -O-alkyl, _-NH2 , alkylamino, Selected from dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic ester, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino C(O)- can be done,
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein said aryl and the heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH ₂ , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, may be substituted with a functional group selected from alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH ₂ , N ₃ , thio, cyclooctyne, -OH, -CO ₂ H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R ₃₂ is selected from Cl, Br, F, mesylate, and tosylate, and R ₃₃ is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-( 4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, R ₃₄ is H, Me, tetrazine-H, and tetrazine-Me;
R ₅ is -CH ₂ OH, -CH ₂ SH, -CH ₂ Cl, -SCH ₂ Cl, -SCH ₂ F, -SCH ₂ CF ₃ , hydroxy, -OCH ₂ CN, -OCH ₂ Cl, -OCH ₂ F, -OCH ₃ , -OCH ₂ CH ₃ , -SCH ₂ CN, and

selected from the group consisting of
R ₆ and R ₇ are independently selected from hydrogen and C _1-10 alkyl;
Q is H,

R ₈ is straight chain or branched alkyl of 1 to 8 carbons, C(O)R ₈ , or n=1 to 4, and R ₄ =H, alkyl or branched alkyl, or P(O)OR ₄ , (C=O)NR ₄ CH _n NR ₄ (C=O)OCH ₂ -(V) _n -J,
A ₁ and A ₂ are independently selected from H and F;
The aforementioned glucocorticoid agonist compounds, in which all possible stereoisomers are claimed, unless otherwise specified. Z is

each of which may be independently substituted with 1 to 4 heteroatoms selected from F, Cl, Br, I, N, S, and O, optionally 1 to 4. further substituted with 4 C _1-3 alkyl or C _1-3 perfluoroalkyl groups,
each of the ring structures may include at least one additional skeletal heteroatom selected from N, S, and O;
During the ceremony, each

indicates a point of attachment to the remainder of the formula, and each of said points of attachment may be covalently bonded to the remainder of the formula through an additional heteroatom selected from N, S, and O. A compound according to item 18. Z-NR ₁ is

selected from
Each of these may be independently substituted with 1 to 4 heteroatoms selected from F, Cl, Br, I, N, S, and O, optionally with 1 to 4 C further substituted with _1-3 alkyl or C _1-3 perfluoroalkyl groups,
each of the ring structures may include at least one additional skeletal heteroatom selected from N, S, and O;
During the ceremony, each

indicates a point of attachment to the remainder of the formula, and each of said points of attachment may be covalently bonded to the remainder of the formula through an additional heteroatom selected from N, S, and O. 20. A compound according to any one of claims or claims 18 or 19. has the structure of formula (II),

During the ceremony,
Y is selected from CH ₂ and O;
E is selected from CH ₂ and O;
G is selected from CH and N;
L is selected from H and F;
R ₅ is -CH ₂ OH, -SCH ₂ F, and

selected from
A ₁ and A ₂ are independently selected from H and F;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein said aryl and the heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH ₂ , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, may be substituted with a functional group selected from alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH ₂ , N ₃ , thio, cyclooctyne, -OH, -CO ₂ H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R ₃₂ is selected from Cl, Br, F, mesylate, and tosylate, and R ₃₃ is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-( 4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R ₃₄ is H, Me, tetrazine-H, and tetrazine-Me. Corticoid agonist compounds. has the structure of formula (III),

During the ceremony,
Y is selected from CH ₂ and O;
E is selected from CH ₂ and O;
G is selected from CH and N;
L is selected from H and F;
R ₅ is -CH ₂ OH, -SCH ₂ F, and

selected from
A ₁ and A ₂ are independently selected from H and F;
V is an alkyl chain of 1 to 8 carbons, a linear or branched polyethylene oxide group containing 1 to 13 units, a linear or branched alkyl group containing 1 to 8 carbons, -O-alkyl, carbon acids, carboxamides, carboxylic acid esters, alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-, 1 to 3 amino acid sequences in which each amino acid independently , Glu, Gly, Asn, Asp, Gln, Leu, Lys, Ala, βAla, Phe, Val, and Cit, aryl, and heteroaryl groups, wherein said aryl and the heteroaryl group is alkyl, haloalkyl, halogen, biphenyl, nitro, nitrile, -OH, -NH ₂ , alkylamino, dialkylamino, thiol, thioalkyl, guanidine, urea, carboxylic acid, alkoxyl, carboxamide, carboxylic acid ester, may be substituted with a functional group selected from alkyl-C(O)O-, alkylamino-C(O)-, dialkylamino-C(O)-,
J is -NH ₂ , N ₃ , thio, cyclooctyne, -OH, -CO ₂ H, transcyclooctene, alkynyl, propargyl,

a reactive group selected from
where R ₃₂ is selected from Cl, Br, F, mesylate, and tosylate, and R ₃₃ is Cl, Br, I, F, OH, -O-N-succinimidyl, -O-( 4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl, and R ₃₄ is H, Me, tetrazine-H, and tetrazine-Me. Corticoid agonist compounds.

23. A compound according to claim 18, 21, or 22 selected from. 23. A compound according to claim 18, 21, or 22, wherein X or Z can be spiro[3.3]heptane or [1.1.1]bicyclopentane and Y can be _CH2 or O. An antibody drug conjugate (ADC) comprising an antibody or antibody-binding fragment thereof, preferably one that binds to an antigen expressed by an immune cell, preferably a human immune cell, wherein the antibody or antibody-binding fragment Said ADC attached to at least one glucocorticoid agonist compound or steroid-linker of any one of the preceding claims or as shown in Figures 118A-O.

selected from
Preferably, where n=2-12, 2-10, 2-8, 2-6, or 2-4, A binds to an antigen expressed by an immune cell, preferably a human immune cell. 26. The ADC according to claim 25, which is an anti-human VISTA antibody, more preferably an anti-human VISTA antibody. Any one of the preceding claims, wherein the linker is selected from any of those disclosed herein or exemplified in the Examples or from the compounds shown in FIGS. 118A-O and FIG. A steroid-linker payload comprising a glucocorticoid agonist as described in Section. PAB and/or amino acids or peptides, optionally 1 to 12 amino acids, more optionally dipeptides, tripeptides, quatrapeptides, pentapeptides, more optionally Gly, Asn, Asp , Gln, Leu, Lys, Ala, Phe, Cit, Val, Val-Cit, Val-Ala, Val-Gly, Val-Gln, Ala-Val, Cit-Cit, Lys-Val-Cit, Asp-Val-Ala , Ala-Ala-Asn, Asp-Val-Ala, Ala-Val-Cit, Ala-Asn-Val, βAla-Leu-Ala-Leu, Lys-Val-Ala, Val-Leu-Lys, Asp-Val-Cit , Val-Ala-Val, and Ala-Ala-Asn, or optionally at least one of GlcA, PAB, and Glu-Gly. A steroid-linker payload comprising a glucocorticoid agonist according to any one of the preceding claims, comprising: At least one glucocorticoid agonist according to any one of the preceding claims, comprising at least one cleavable linker and/or sacrificial linker attached directly or indirectly to the glucocorticoid agonist steroid compound. Steroid-linker payload containing compound. An antibody-drug conjugate (ADC) comprising an antibody or antibody-binding fragment thereof, preferably one that binds to an antigen expressed by an immune cell, preferably an antigen expressed on a human immune cell, wherein the antibody or said ADC, wherein the antibody binding fragment is attached to at least one glucocorticoid agonist or steroid-linker compound according to any one of the preceding claims.

selected from
Preferably, where n=2-12, 2-10, 2-8, 2-6, or 2-4, A binds to an antigen expressed by an immune cell, preferably a human immune cell. 31. The ADC according to claim 30, which is an anti-human VISTA antibody, more preferably an anti-human VISTA antibody. A composition comprising at least one glucocorticoid agonist compound or steroid linker conjugate or ADC according to any one of the preceding claims and a pharmaceutically acceptable carrier. 33. The composition of claim 32, which is suitable for in vivo administration to a subject in need thereof. 34. A composition according to claim 32 or 33, comprising at least one additive. 35. The composition of claim 32, 33, or 34, comprising at least one stabilizer or buffer. A composition according to any one of the preceding claims, optionally suitable for parenteral administration by injection. Any of the preceding claims is suitable for injection into a subject in need thereof, optionally via intravenous, subcutaneous, intramuscular, intratumoral, intranodal, intranasal, or intrathecal injection. The composition according to item 1. A composition according to any one of the preceding claims, which is administrable subcutaneously. According to any one of the preceding claims, comprised in a device for providing subcutaneous administration selected from the group consisting of a syringe, an injection device, an infusion pump, an injector pen, a needleless device, an auto-injector, and a subcutaneous patch delivery system. Composition of. 40. The device of claim 39, wherein the device delivers a fixed dose of glucocorticoid receptor agonist to the patient. A glucocorticoid agonist compound or steroid linker conjugate or ADC according to any one of the preceding claims or for the treatment, prevention or inhibition of inflammation or autoimmunity in a subject in need thereof. Use of compositions containing. Glucocorticoid agonist compounds or steroid linker conjugates or ADCs according to any one of the preceding claims, or in a subject in need of such treatment, prophylaxis, or inhibition of inflammatory or autoimmune or allergic reactions. A composition containing for use in the preparation of a medicament for. A method of treatment and/or prophylaxis comprising at least one glucocorticoid agonist compound or steroid linker conjugate or ADC according to any one of the preceding claims, or comprising at least one glucocorticoid agonist compound or steroid linker conjugate or ADC according to any one of the preceding claims. The method comprises administering a composition to a subject in need of treatment and/or prevention. In human subjects, for the treatment of allergy, autoimmunity, transplantation, gene therapy, inflammation, GVHD, or sepsis, or to treat inflammatory, autoimmune, or allergic side effects associated with any of the aforementioned conditions. A use, medicament, composition or method according to any one of the preceding claims, for or for the prophylaxis. A use, medicament, composition or method according to any one of the preceding claims, which is for acute use. A use, medicament, composition or method according to any one of the preceding claims, which is for chronic use. Use, medicament, composition or method according to any one of the preceding claims for maintenance therapy. for the treatment or prevention of acute or chronic inflammation and the autoimmune and inflammatory indications associated therewith, optionally said conditions including acquired aplastic anemia+, acquired hemophilia +, acute disseminated encephalomyelitis (ADEM) +, acute hemorrhagic leukoencephalitis (AHLE)/Hurst's disease +, primary agammaglobulinemia +, alopecia areata +, ankylosing spondylitis (AS), anti-NMDA Receptor encephalitis+, antiphospholipid syndrome (APS)+, arteriosclerosis, autism spectrum disorder (ASD), autoimmune Addison's disease (AAD)+, autoimmune autonomic neuropathy/autoimmune autonomic ganglion disorder (AAG), autoimmune encephalitis+, autoimmune gastritis, autoimmune hemolytic anemia (AIHA)+, autoimmune hepatitis (AIH)+, autoimmune hyperlipidemia, autoimmune hypophysitis/ Lymphocytic hypophysitis+, autoimmune inner ear disease (AIED)+, autoimmune lymphoproliferative syndrome (ALPS)+, autoimmune myocarditis, autoimmune oophoritis+, autoimmune orchitis+, autoimmune Immune pancreatitis (AIP)/immunoglobulin G4-related disease (IgG4-RD)+, type I, type II, and type III autoimmune polyglandular syndrome+, autoimmune progesterone dermatitis+, autoimmune sudden deafness (SNHL), atonia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema , autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune Pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy (AMAN), Baro's disease, Behcet's disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease disease, Chagas disease, chronic inflammatory demyelinating polyneuritis (CIDP), chronic relapsing polyosteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial Pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, type 1 diabetes, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrotic lung disease cystitis, fibrosing alveolitis, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch's Schonlein purpura (HSP), gestational herpes or pemphigoid (PG), sweat gland abscess (HS) (opposite acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immunity Thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert's・Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (including nephritis and skin), chronic Lyme disease, Meniere's disease, microscopic polyvascular disease inflammatory syndrome (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucka-Habelmann disease, multiple motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasia gravis, myelin oligodendrocyte glycoprotein Antibody disorders, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, opsoclonus-myoclonus syndrome (OMS), rheumatoid arthritis (PR), PANDAS, paraneoplastic Cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars plana (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis , pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, type I, II, III polyglandular syndrome, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cholangitis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, erythroblastic aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy , relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, Stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), thyroid eye disease (TED), Tolosa Hunt syndrome (THS), transverse myelitis, type 1 diabetes, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease A use, medicament, composition or method according to any one of the preceding claims, comprising: for the treatment or prevention of acute or chronic inflammation and associated autoimmune and inflammatory and allergic indications or side effects, said conditions optionally including severe asthma, giant cell arteritis, ANKA Use, medicament, composition or method according to any one of the preceding claims, including vasculitis and IBD (colitis and Crohn's disease). Rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, sweat gland abscess, uveitis, Behcet's disease, spondyloarthropathy, or Use, medicament, composition or method according to any one of the preceding claims for the treatment or prevention of a condition selected from psoriasis. One or more of the following:
(i) a chronic, acute, episodic allergy, inflammation, or inflammatory condition, e.g., chronic, acute, episodic, remitting/relapsing;
(ii) a condition that can be effectively treated only temporarily with high doses of steroids, optionally for which the patient is being treated or is being treated with high doses of steroids; polymyalgia and/or giant cell arteritis,
(iii) Conditions with comorbidities that limit steroid use, optionally diabetes, nonalcoholic steatohepatitis (NASH), morbid obesity, avascular necrosis/osteonecrosis (AVN), glaucoma, steroid-induced high blood pressure, severe skin fragility, and/or osteoarthritis;
(iv) conditions for which safe long-term therapeutic agents are available, but where induction for several months with high doses of steroids is desired; optionally, induction for several months with high doses of steroids is therapeutic; AAV, polymyositis, dermatomyositis, lupus, inflammatory lung disease, autoimmune hepatitis, inflammatory bowel disease, immune thrombocytopenia, autoimmune hemolytic anemia, gout patients,
(v) skin conditions requiring short-term/long-term treatment, optionally conditions of uncertain treatment or duration, and/or conditions for which there are no effective alternatives to steroid administration, optionally Stevens Johnson, other severe drug eruption conditions, conditions with extensive contact dermatitis, other severe immune-related skin conditions such as PG, LCV, erythroderma, and the like;
(vi) conditions treated with high-dose corticosteroids for flare-ups/relapses, optionally COPD, asthma, lupus, gout, pseudogout;
(vii) immune-related neurological diseases such as small fiber neuropathy, MS (subset), chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, and the like;
(viii) optionally hematology/oncology indications for which high doses of steroids are therapeutically warranted or beneficial;
(ix) ophthalmological conditions, optionally uveitis, iritis, scleritis, and the like;
(x) conditions associated with persistent or very long-term adrenal insufficiency or secondary adrenal insufficiency, optionally an iatrogenic Addison's disease crisis;
(xi) A prior claim that is for treatment or prophylaxis in patients with conditions often treated with long-term, low-dose steroids, optionally lupus, RA, psA, vasculitis, and the like. A use, medicament, composition, or method according to any one of paragraphs. For treatment or prophylaxis in special classes of patients at risk of toxicity on steroid therapy, such as pregnant/lactating women, pediatric patients, and optionally pediatric patients with growth defects or cataracts. The use, medicament, composition or method of any one of the preceding claims, wherein the patient is further treated with another active agent. The patient has an immunoinhibitory antibody or fusion protein targeting one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA; and/or further treated with an immunomodulatory antibody or fusion protein selected from agonist antibodies or fusion proteins targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28, or ICOS. A use or method according to any one of the claims. said ADC comprises an antibody or antigen-binding fragment ("A") comprising an antigen-binding region that specifically binds to human V-domain Ig inhibitor of T cell activation (human VISTA); When administered to a subject, VISTA-expressing immune cells, optionally monocytes, myeloid cells, T cells, Tregs, NK cells, neutrophils, dendritic cells, macrophages, eosinophils, and endothelial cells. The antibody of any one of the preceding claims, which is preferentially delivered to one or more of the immune cells, resulting in functional internalization of the anti-inflammatory agent into one or more of the immune cells. Drug conjugates (ADCs), uses, agents, compositions, or methods. an anti-human VISTA antibody or antibody that preferentially binds to VISTA-expressing cells at physiological pH (≈7.5), wherein said ADC optionally has a pK of at most 70 hours in human VISTA knock-in rodents; An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims, comprising a fragment. The ADC binds to VISTA-expressing cells at physiological pH and in cynomolgus monkeys or humans at physiological pH for up to 3.5 ± 0.5 days, more typically for up to 48 hours, for up to 24 hours. An antibody drug conjugate (ADC), use, medicament, according to any one of the preceding claims, comprising an anti-human VISTA antibody or antibody fragment having a pK of at most 18 hours, or at most 12 hours; Composition or method. The anti-human ADC has a pK of at most 2.8 or 2.3 or 1.5 days, or 1 day, or 12 hours or 8 hours ± 0.5 days in cynomolgus monkeys or humans at physiological pH. An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims, comprising a VISTA antibody or antibody fragment. An antibody drug conjugate according to any one of the preceding claims, wherein the ADC comprises an anti-human VISTA antibody or antibody fragment having a pK of at most 6-12 hours in human VISTA rodents at physiological pH. (ADC), use, medicament, composition, or method. said ADC is a VISTA-expressing immune cell, optionally one of a T cell, a Treg, a NK cell, a neutrophil, a monocyte, a myeloid cell, a dendritic cell, a macrophage, an eosinophil, and an endothelial cell. an anti-human VISTA antibody comprising a linker that is cleaved upon internalization of said ADC into said immune cells, resulting in the release of a therapeutically effective amount of an anti-inflammatory agent within said immune cells, which induces anti-inflammatory activity; or an antibody fragment. The ADC comprises an anti-human VISTA antibody or antibody fragment, and the anti-VISTA antibody or antigen-binding fragment has a pH of about 2.3 in a primate, optionally a cynomolgus monkey, at physiological pH (about pH 7.5). An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims, having an in vivo serum half-life of days. The ADC comprises an anti-human VISTA antibody or antibody fragment, and the anti-VISTA antibody or antigen-binding fragment is present at physiological pH (approximately pH 7.5) in human VISTA knock-in rodents for up to 70 hours, up to 60 hours, 50 hours or less, 40 hours or less, 30 hours or less, 24 hours or less, 22-24 hours or less, 20-22 hours or less, 18-20 hours or less, 16-18 hours or less, 14-16 hours or less, 12-14 hours 10 to 12 hours or less, 8 to 10 hours or less, 6 to 8 hours or less, 4 to 6 hours or less, 2 to 4 hours or less, 1 to 2 hours or less, 0.5 to 1.0 hours or less, or 0 An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims, having an in vivo serum half-life in serum of .1 to 0.5 hours or less. When the ADC comprises an anti-human VISTA antibody or antibody fragment, and the PD/PK ratio of the ADC is used in vivo, a human VISTA knock-in rodent, or a human or non-human primate, optionally, In cynomolgus monkeys, at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13: 1, 14:1, 28:1, or higher. 1, 14:1, 28:1, or more. said ADC comprises an anti-human VISTA antibody or antibody fragment, and said ADC has a PD of at least 4 in any one of a rodent, or a human or non-human primate, optionally a cynomolgus monkey; 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 28 days, 2 to 3 weeks, 1 month, 2 months, or longer to any one of the preceding claims. Antibody drug conjugates (ADCs), uses, medicaments, compositions, or methods described. The antibody drug according to any one of the preceding claims, wherein the ADC comprises an anti-human VISTA antibody or antibody fragment, and wherein the anti-human VISTA antibody comprises an Fc region with impaired or intact FcR binding. Conjugates (ADCs), uses, agents, compositions, or methods. The ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, and the antibody or antibody fragment comprises a human IgG1, IgG2, IgG3, or human IgG1 with impaired or intact FcR binding. An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims, comprising an IgG4 Fc region. Any of the preceding claims, wherein said ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, said antibody or antibody fragment comprising a human IgG1 Fc region with impaired FcR binding. An antibody drug conjugate (ADC), use, medicament, composition, or method according to paragraph 1. An antibody or antibody fragment, optionally, wherein said ADC comprises a human or non-human primate constant or Fc region that has been modified to impair or eliminate binding to at least two naturally occurring human Fc gamma receptors. , anti-human VISTA antibody or antibody fragment. The ADC is any one, two, three, or four of the following FcRs: hFcγRI (CD64), FcyRIIA or hFcyRIIB, (CD32 or CD32A), and FcγRllA (CD16A) or FcγRllB (CD16B), or an antibody or antibody fragment comprising a human or non-human primate constant or Fc region that has been modified to impair or eliminate binding to all five, optionally an anti-human VISTA antibody or antibody fragment. , an antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims. An antibody or antibody fragment, wherein said ADC comprises a human IgG2 kappa backbone, optionally with a V234A/G237A/P238S/H268A/V309L/A330S/P331S silencing mutation in said Fc region; An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims, comprising an anti-human VISTA antibody or antibody fragment. The ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, optionally an L234A/L235A silencing mutation in the Fc region, optionally a complement (C1 _Q ) An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims, comprising an IgG1/kappa backbone with a mutation that impairs binding. An antibody or antibody fragment, optionally an anti-human VISTA antibody, wherein said ADC comprises a human IgG1/kappa backbone, optionally having L234A/L235A silencing mutations and E269R and E233A mutations in said Fc region. or an antibody fragment. Any of the preceding claims, wherein the ADC comprises an anti-human VISTA antibody or antibody fragment, and the binding of the anti-VISTA antibody or antigen-binding fragment to VISTA-expressing immune cells does not directly agonize or antagonize VISTA-mediated effects on immunity. An antibody drug conjugate (ADC), use, medicament, composition, or method according to item 1. The preceding claim wherein said ADC comprises an antibody or antibody fragment comprising a human IgG1, IgG2, IgG3, or IgG4 Fc region, optionally an anti-human VISTA antibody or antibody fragment, and endogenous FcR binding is not impaired. An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of paragraphs. An antibody-drug conjugate according to any one of the preceding claims, wherein said ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, comprising a native (unmodified) human IgG2 Fc region. Gate (ADC), use, agent, composition, or method. said ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, said antibody or antigen-binding fragment as determined by surface plasmon resonance (SPR) at 24°C or 37°C. An antibody drug conjugate (ADC) according to any one of the preceding claims, comprising a KD in the range of .0001 nM to 10.0 nM, 0.001 to 1.0 nM, or 0.01 to 0.7; Uses, agents, compositions, or methods. The ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, wherein the antibody or antigen-binding fragment is at 0.001 mV as determined by surface plasmon resonance (SPR) at 24°C or 37°C. An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims, comprising a KD of 13 to 0.64 nM. of the preceding claims, wherein the ADC optionally comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, and the drug-antibody ratio is in the range of 1:1 to 12:1. An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding paragraphs. The ADC optionally comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, and the drug-antibody ratio is 2-12:1, 2-8:1, 4-8 :1, or in the range of 6 to 8:1. the ADC optionally comprises an anti-human VISTA antibody or antibody fragment, and the drug-antibody ratio is about 8:1 (n=8) or about 4:1 (n=4) , an antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims. The ADC is one or more of monocytes, myeloid cells, T cells, Tregs, CD4 T cells, CD8 T cells, macrophages, NK cells, macrophages, eosinophils, mast cells, B cells, and neutrophils. An antibody drug conjugate (ADC), use, medicament, composition according to any one of the preceding claims, comprising an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, for internalizing , or method. An antibody-drug conjugate (ADC) according to any one of the preceding claims, wherein said ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, that does not obviously internalize B cells. ), uses, agents, compositions, or methods. When the ADC is administered to a subject in need thereof, the ADC promotes efficacy and/or improves the effectiveness of the anti-inflammatory agent when administered to a subject in need thereof, compared to the same dose of anti-inflammatory agent administered in naked (unconjugated) form. An antibody-drug conjugate according to any one of the preceding claims, comprising an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, which reduces adverse side effects such as toxicity associated with inflammatory agents. (ADC), use, medicament, composition, or method. said ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, and said glucocorticoid is optionally conjugated to said antibody or antigen-binding fragment via an interchain disulfide. An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims. Antibody drug conjugate (ADC) according to any one of the preceding claims, use, wherein said ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, comprising an esterase sensitive linker. , agent, composition, or method. The ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, and the cleavable linker is capable of acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage. An antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims, which is susceptible to one or more of the following: The ADC comprises an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment, and the antigen-binding fragment contained in the ADC comprises a Fab, F(ab')2, or scFv antibody fragment. , an antibody drug conjugate (ADC), use, medicament, composition, or method according to any one of the preceding claims. the ADC comprises an anti-human VISTA antibody or antibody fragment, wherein the anti-VISTA antibody or antibody fragment contained therein comprises the same CDRs as an antibody having the sequence of FIG. 8, 10, or 12, or optionally In particular,
(i) comprising the V _H CDRs of SEQ ID NOs: 100, 101, and 102 and the V _L CDRs of SEQ ID NOs: 103, 104, and 105;
(ii) comprising the V _H CDRs of SEQ ID NOs: 110, 111, and 112 and the V _L CDRs of SEQ ID NOs: 113, 114, and 115;
(iii) comprising the V _H CDRs of SEQ ID NOs: 120, 121, and 122 and the V _L CDRs of SEQ ID NOs: 123, 124, and 125;
(iv) comprising the V _H CDRs of SEQ ID NOs: 130, 131, and 132 and the V _L CDRs of SEQ ID NOs: 133, 134, and 135;
(v) comprising the V _H CDRs of SEQ ID NOs: 140, 141, and 142 and the V _L CDRs of SEQ ID NOs: 143, 144, and 145;
(vi) comprising the V _H CDRs of SEQ ID NOs: 150, 151, and 152 and the V _L CDRs of SEQ ID NOs: 153, 154, and 155;
(vii) comprising the V _H CDRs of SEQ ID NOs: 160, 161, and 162 and the V _L CDRs of SEQ ID NOs: 163, 164, and 165;
(viii) comprising the V _H CDRs of SEQ ID NOs: 170, 171, and 172 and the V _L CDRs of SEQ ID NOs: 173, 174, and 175;
(ix) those comprising the V _H CDRs of SEQ ID NOs: 180, 181, and 182 and the V _L CDRs of SEQ ID NOs: 183, 184, and 185;
(x) comprising the V _H CDRs of SEQ ID NOs: 190, 191, and 192 and the V _L CDRs of SEQ ID NOs: 193, 194, and 195;
(xi) comprising the V _H CDRs of SEQ ID NOs: 200, 201, and 202 and the V _L CDRs of SEQ ID NOs: 203, 204, and 205;
(xii) comprising the V _H CDRs of SEQ ID NOs: 210, 211, and 212 and the V _L CDRs of SEQ ID NOs: 213, 214, and 215;
(xiii) those comprising the V _H CDRs of SEQ ID NOs: 220, 221, and 222 and the V _L CDRs of SEQ ID NOs: 223, 224, and 225;
(xiv) those comprising the V _H CDRs of SEQ ID NOs: 230, 231, and 232 and the V _L CDRs of SEQ ID NOs: 233, 234, and 235;
(xv) those comprising the V _H CDRs of SEQ ID NOs: 240, 241, and 242 and the V _L CDRs of SEQ ID NOs: 243, 244, and 245;
(xvi) those comprising the V _H CDRs of SEQ ID NOs: 250, 251, and 252 and the V _L CDRs of SEQ ID NOs: 253, 254, and 255;
(xvii) comprising the VH CDRs of SEQ ID NOs: 260, 261, and 262 and the V _L CDRs of SEQ ID NOs: 263, 264, and 265;
(xviii) those comprising the V _H CDRs of SEQ ID NOs: 270, 271, and 272 and the V _L CDRs of SEQ ID NOs: 273, 274, and 275;
(xix) those comprising the V _H CDRs of SEQ ID NOs: 280, 281, and 282 and the V _L CDRs of SEQ ID NOs: 283, 284, and 285;
(xx) those comprising the V _H CDRs of SEQ ID NOs: 290, 291, and 292 and the V _L CDRs of SEQ ID NOs: 293, 294, and 295;
(xxi) those comprising the V _H CDRs of SEQ ID NOs: 300, 301, and 302 and the V _L CDRs of SEQ ID NOs: 303, 304, and 305;
(xxii) comprising the V _H CDRs of SEQ ID NOs: 310, 311, and 312 and the V _L CDRs of SEQ ID NOs: 313, 314, and 315;
(xxiii) comprising the V _H CDRs of SEQ ID NOs: 320, 321, and 322 and the V _L CDRs of SEQ ID NOs: 323, 324, and 325;
(xxiv) those comprising the V _H CDRs of SEQ ID NOs: 330, 331, and 332 and the V _L CDRs of SEQ ID NOs: 333, 334, and 335;
(xxv) those comprising the V _H CDRs of SEQ ID NOs: 340, 341, and 342 and the V _L CDRs of SEQ ID NOs: 343, 344, and 345;
(xxvi) those comprising the V _H CDRs of SEQ ID NOs: 350, 351, and 352 and the V _L CDRs of SEQ ID NOs: 353, 354, and 355;
(xxvii) those comprising the V _H CDRs of SEQ ID NOs: 360, 361, and 362 and the V _L CDRs of SEQ ID NOs: 363, 364, and 365;
(xxviii) comprising the V _H CDRs of SEQ ID NOs: 370, 371, and 372 and the V _L CDRs of SEQ ID NOs: 373, 374, and 375;
(xxix) comprising the V _H CDRs of SEQ ID NOs: 380, 381, and 382 and the V _L CDRs of SEQ ID NOs: 383, 384, and 385;
(xxx) comprising the V _H CDRs of SEQ ID NOs: 390, 391, and 392 and the V _L CDRs of SEQ ID NOs: 393, 394, and 395;
(xxxi) those comprising the V _H CDRs of SEQ ID NOs: 400, 401, and 402 and the V _L CDRs of SEQ ID NOs: 403, 404, and 405;
(xxxii) comprising the V _H CDRs of SEQ ID NOs: 410, 411, and 412 and the V _L CDRs of SEQ ID NOs: 413, 414, and 415;
(xxxiii) those comprising the V _H CDRs of SEQ ID NOs: 420, 421, and 422 and the V _L CDRs of SEQ ID NOs: 423, 424, and 425;
(xxxiv) those comprising the V _H CDRs of SEQ ID NOs: 430, 431, and 432 and the V _L CDRs of SEQ ID NOs: 433, 434, and 435;
(xxxv) those comprising the V _H CDRs of SEQ ID NOs: 440, 441, and 442 and the V _L CDRs of SEQ ID NOs: 443, 444, and 445;
(xxxvi) those comprising the V _H CDRs of SEQ ID NOs: 450, 451, and 452 and the V _L CDRs of SEQ ID NOs: 453, 454, and 455;
(xxxvii) comprising the V _H CDRs of SEQ ID NOs: 460, 461, and 462 and the V _L CDRs of SEQ ID NOs: 463, 464, and 465;
(xxxviii) comprising the V _H CDRs of SEQ ID NOs: 470, 471, and 472 and the V _L CDRs of SEQ ID NOs: 473, 474, and 475;
(xxxix) comprising the V _H CDRs of SEQ ID NOs: 480, 481, and 482 and the V _L CDRs of SEQ ID NOs: 483, 484, and 485;
(xl) those comprising the V _H CDR polypeptides of SEQ ID NOs: 490, 491, and 492 and the VL CDR polypeptides of SEQ ID NOs: 493, 494, and 495;
(xli) those comprising the V _H CDR polypeptides of SEQ ID NOs: 500, 501, and 502 and the VL CDR polypeptides of SEQ ID NOs: 503, 504, and 505;
(xlii) comprising the V _H CDR polypeptides of SEQ ID NOs: 510, 511, and 512 and the VL CDR polypeptides of SEQ ID NOs: 513, 514, and 515;
(xliii) comprising the V _H CDR polypeptides of SEQ ID NOs: 520, 521, and 522 and the VL CDR polypeptides of SEQ ID NOs: 523, 524, and 525;
(xliv) comprising the V _H CDR polypeptides of SEQ ID NOs: 530, 531, and 532, and the VL CDR polypeptides of SEQ ID NOs: 533, 534, and 535;
(xlv) comprising the V _H CDR polypeptides of SEQ ID NOs: 540, 541, and 542, and the VL CDR polypeptides of SEQ ID NOs: 543, 544, and 545;
(xlvi) comprising the V _H CDR polypeptides of SEQ ID NOs: 550, 551, and 552, and the VL CDR polypeptides of SEQ ID NOs: 553, 554, and 555;
(xlvii) those comprising the V _H CDRs of SEQ ID NOs: 560, 561, and 562 and the V _L CDRs of SEQ ID NOs: 563, 564, and 565;
(xlviii) those comprising the V _H CDRs of SEQ ID NOs: 570, 571, and 572 and the V _L CDRs of SEQ ID NOs: 573, 574, and 575;
(xlix) comprising the V _H CDRs of SEQ ID NOs: 580, 581, and 582 and the V _L CDRs of SEQ ID NOs: 583, 584, and 585;
(l) comprising the V _H CDRs of SEQ ID NOs: 590, 591, and 592 and the V _L CDRs of SEQ ID NOs: 593, 594, and 595;
(li) comprising the V _H CDRs of SEQ ID NOs: 600, 601, and 602 and the V _L CDRs of SEQ ID NOs: 603, 604, and 605;
(lii) comprising the V _H CDRs of SEQ ID NOs: 610, 611, and 612 and the V _L CDRs of SEQ ID NOs: 613, 614, and 615;
(liii) those comprising the V _H CDRs of SEQ ID NOs: 620, 621, and 622 and the V _L CDRs of SEQ ID NOs: 623, 624, and 625;
(liv) those comprising the V _H CDRs of SEQ ID NOs: 630, 631, and 632 and the V _L CDRs of SEQ ID NOs: 633, 634, and 635;
(lv) those comprising the V _H CDRs of SEQ ID NOs: 640, 641, and 642 and the V _L CDRs of SEQ ID NOs: 643, 644, and 645;
(lvi) those comprising the V _H CDRs of SEQ ID NOs: 650, 651, and 652 and the V _L CDRs of SEQ ID NOs: 653, 654, and 655;
(lvii) comprising the V _H CDRs of SEQ ID NOs: 660, 661, and 662 and the V _L CDRs of SEQ ID NOs: 663, 664, and 665;
(lviii) those comprising the V _H CDRs of SEQ ID NOs: 670, 671, and 672 and the V _L CDRs of SEQ ID NOs: 673, 674, and 675;
(lix) those comprising the V _H CDRs of SEQ ID NOs: 680, 681, and 682 and the V _L CDRs of SEQ ID NOs: 683, 684, and 685;
(lx) comprising the V _H CDRs of SEQ ID NOs: 690, 691, and 692 and the V _L CDRs of SEQ ID NOs: 693, 694, and 695;
(lxi) those comprising the V _H CDRs of SEQ ID NOs: 700, 701, and 702 and the V _L CDRs of SEQ ID NOs: 703, 704, and 705;
(lxii) comprising the V _H CDRs of SEQ ID NOs: 710, 711, and 712 and the V _L CDRs of SEQ ID NOs: 713, 714, and 715;
(lxiii) those comprising the V _H CDRs of SEQ ID NOs: 720, 721, and 722 and the V _L CDRs of SEQ ID NOs: 723, 724, and 725;
(lxiv) those comprising the V _H CDRs of SEQ ID NOs: 730, 731, and 732 and the V _L CDRs of SEQ ID NOs: 733, 734, and 735;
(lxv) comprising the V _H CDRs of SEQ ID NOs: 740, 741, and 742 and the V _L CDRs of SEQ ID NOs: 743, 744, and 745;
(lxvi) comprising the V _H CDRs of SEQ ID NOs: 750, 751, and 752 and the V _L CDRs of SEQ ID NOs: 753, 754, and 755;
(lxvii) comprising the V _H CDRs of SEQ ID NOs: 760, 761, and 762 and the V _L CDRs of SEQ ID NOs: 763, 764, and 765;
(lxviii) those comprising the V _H CDRs of SEQ ID NOs: 770, 771, and 772 and the V _L CDRs of SEQ ID NOs: 773, 774, and 775;
(lxix) comprising the V _H CDRs of SEQ ID NOs: 780, 781, and 782 and the V _L CDRs of SEQ ID NOs: 783, 784, and 785;
(lxx) comprising the V _H CDRs of SEQ ID NOs: 790, 791, and 792 and the V _L CDRs of SEQ ID NOs: 793, 794, and 795;
(lxxi) those comprising the V _H CDRs of SEQ ID NOs: 800, 801, and 802 and the V _L CDRs of SEQ ID NOs: 803, 804, and 805;
(lxxii) the V _H CDRs of SEQ ID NOs: 810, 811, and 812; and the V _L CDRs of SEQ ID NOs: 813, 814, and 815; Antibody drug conjugates (ADCs), uses, medicaments, compositions, or methods described. The antibody drug conjugate of any one of the preceding claims, wherein the ADC comprises an anti-VISTA antibody or antibody fragment comprising the same CDR as any one of VSTB92, VSTB56, VSTB95, VSTB103, and VSTB66. (ADC). The ADC comprises an anti-VISTA antibody or antibody fragment comprising a V _H polypeptide and _a V _L polypeptide, each of _which comprises:
(i) comprising a V _H polypeptide of SEQ ID NO: 106 identity and a V _L polypeptide of SEQ ID NO: 108;
(ii) comprising a V _H polypeptide of SEQ ID NO: 116 and a V _L polypeptide of SEQ ID NO: 118;
(iii) comprising a V _H polypeptide of SEQ ID NO: 126 and a V _L polypeptide of SEQ ID NO: 128;
(iv) comprising a V _H polypeptide of SEQ ID NO: 136 and a V _L polypeptide of SEQ ID NO: 138;
(v) comprising a V _H polypeptide of SEQ ID NO: 146 and a V _L polypeptide of SEQ ID NO: 148;
(vi) comprising a V _H polypeptide of SEQ ID NO: 156 and a V _L polypeptide of SEQ ID NO: 158;
(vii) comprising a V _H polypeptide of SEQ ID NO: 166 and a V _L polypeptide of SEQ ID NO: 168;
(viii) comprising a V _H polypeptide of SEQ ID NO: 176 and a V _L polypeptide of SEQ ID NO: 178;
(ix) comprising the V _H polypeptide of SEQ ID NO: 186 and the V _L polypeptide of SEQ ID NO: 188;
(x) one comprising the V _H polypeptide of SEQ ID NO: 196 and the V _L polypeptide of SEQ ID NO: 198;
(xi) comprising the V _H polypeptide of SEQ ID NO: 206 and the V _L polypeptide of SEQ ID NO: 208;
(xii) comprising the V _H polypeptide of SEQ ID NO: 216 and the V _L polypeptide of SEQ ID NO: 218;
(xiii) comprising a V _H polypeptide of SEQ ID NO: 226 and a V _L polypeptide of SEQ ID NO: 228;
(xiv) comprising a V _H polypeptide of SEQ ID NO: 236 and a V _L polypeptide of SEQ ID NO: 238;
(xv) comprising a V _H polypeptide of SEQ ID NO: 246 and a V _L polypeptide of SEQ ID NO: 248;
(xvi) comprising a V _H polypeptide of SEQ ID NO: 256 and a V _L polypeptide of SEQ ID NO: 258;
(xvii) comprising a V _H polypeptide of SEQ ID NO: 266 and a V _L polypeptide of SEQ ID NO: 268;
(xviii) comprising a V _H polypeptide of SEQ ID NO: 276 and a VL polypeptide of SEQ ID NO: 278;
(xix) comprising a V _H polypeptide of SEQ ID NO: 286 and a V _L polypeptide of SEQ ID NO: 288;
(xx) comprising a V _H polypeptide of SEQ ID NO: 296 and a V _L polypeptide of SEQ ID NO: 298;
(xxi) one comprising a V _H polypeptide of SEQ ID NO: 306 and a V _L polypeptide of SEQ ID NO: 308;
(xxii) comprising a V _H polypeptide of SEQ ID NO: 316 and a V _L polypeptide of SEQ ID NO: 318;
(xxiii) comprising a V _H polypeptide of SEQ ID NO: 326 and a V _L polypeptide of SEQ ID NO: 328;
(xxiv) comprising a V _H polypeptide of SEQ ID NO: 336 and a V _L polypeptide of SEQ ID NO: 338;
(xxv) comprising a V _H polypeptide of SEQ ID NO: 346 and a V _L polypeptide of SEQ ID NO: 348;
(xxvi) comprising a V _H polypeptide of SEQ ID NO: 356 and a V _L polypeptide of SEQ ID NO: 358;
(xxvii) comprising a V _H polypeptide of SEQ ID NO: 366 and a V _L polypeptide of SEQ ID NO: 368;
(xxviii) comprising a V _H polypeptide of SEQ ID NO: 376 and a V _L polypeptide of SEQ ID NO: 378;
(xxix) comprising a V _H polypeptide of SEQ ID NO: 386 and a V _L polypeptide of SEQ ID NO: 388;
(xxx) comprising a V _H polypeptide of SEQ ID NO: 396 and a V _L polypeptide of SEQ ID NO: 398;
(xxxi) comprising a V _H polypeptide of SEQ ID NO: 406 and a V _L polypeptide of SEQ ID NO: 408;
(xxxii) comprising a V _H polypeptide of SEQ ID NO: 416 and a V _L polypeptide of SEQ ID NO: 418;
(xxxiii) comprising a V _H polypeptide of SEQ ID NO: 426 and a V _L polypeptide of SEQ ID NO: 428;
(xxxiv) comprising a V _H polypeptide of SEQ ID NO: 436 and a V _L polypeptide of SEQ ID NO: 438;
(xxxv) comprising a V _H polypeptide of SEQ ID NO: 446 and a V _L polypeptide of SEQ ID NO: 448;
(xxxvi) comprising a V _H polypeptide of SEQ ID NO: 456 and a V _L polypeptide of SEQ ID NO: 458;
(xxxvii) comprising a V _H polypeptide of SEQ ID NO: 466 and a V _L polypeptide of SEQ ID NO: 468;
(xxxviii) comprising a V _H polypeptide of SEQ ID NO: 476 and a V _L polypeptide of SEQ ID NO: 478;
(xxxix) comprising a V _H polypeptide of SEQ ID NO: 486 and a V _L polypeptide of SEQ ID NO: 488;
(xl) comprising a V _H polypeptide of SEQ ID NO: 496 and a V _L polypeptide of SEQ ID NO: 498;
(xli) one comprising a V _H polypeptide of SEQ ID NO: 506 and a V _L polypeptide of SEQ ID NO: 508;
(xlii) comprising a V _H polypeptide of SEQ ID NO: 516 and a V _L polypeptide of SEQ ID NO: 518;
(xliii) comprising a V _H polypeptide of SEQ ID NO: 526 and a V _L polypeptide of SEQ ID NO: 528;
(xliv) comprising a V _H polypeptide of SEQ ID NO: 536 and a V _L polypeptide of SEQ ID NO: 533, 534, and 535;
(xlv) comprising a V _H polypeptide of SEQ ID NO: 546 and a V _L polypeptide of SEQ ID NO: 548;
(xlvi) comprising a V _H polypeptide of SEQ ID NO: 556 and a V _L polypeptide of SEQ ID NO: 558;
(xlvii) comprising a V _H polypeptide of SEQ ID NO: 566 and a V _L polypeptide of SEQ ID NO: 568;
(xlviii) comprising a V _H polypeptide of SEQ ID NO: 576 and a V _L polypeptide of SEQ ID NO: 578;
(xlix) comprising a V _H polypeptide of SEQ ID NO: 586 and a V _L polypeptide of SEQ ID NO: 588;
(l) comprising a V _H polypeptide of SEQ ID NO: 596 and a V _L polypeptide of SEQ ID NO: 598;
(li) comprising a V _H polypeptide of SEQ ID NO: 606 and a V _L polypeptide of SEQ ID NO: 608;
(lii) comprising a V _H polypeptide of SEQ ID NO: 616 and a V _L polypeptide of SEQ ID NO: 618;
(liii) comprising a V _H polypeptide of SEQ ID NO: 626 and a V _L polypeptide of SEQ ID NO: 628;
(liv) comprising the V _H polypeptide of SEQ ID NO: 636 and the V _L polypeptide of SEQ ID NO: 638;
(lv) comprising a V _H polypeptide of SEQ ID NO: 646 and a V _L polypeptide of SEQ ID NO: 648;
(lvi) comprising a V _H polypeptide of SEQ ID NO: 656 and a V _L polypeptide of SEQ ID NO: 658;
(lvii) comprising a V _H polypeptide of SEQ ID NO: 666 and a V _L polypeptide of SEQ ID NO: 668;
(lviii) comprising a V _H polypeptide of SEQ ID NO: 676 and a V _L polypeptide of SEQ ID NO: 678;
(lix) comprising a V _H polypeptide of SEQ ID NO: 686 and a V _L polypeptide of SEQ ID NO: 688;
(lx) comprising a V _H polypeptide of SEQ ID NO: 696 and a V _L polypeptide of SEQ ID NO: 698;
(lxi) one comprising a V _H polypeptide of SEQ ID NO: 706 and a V _L polypeptide of SEQ ID NO: 708;
(lxii) comprising a V _H polypeptide of SEQ ID NO: 716 and a V _L polypeptide of SEQ ID NO: 718;
(lxiii) comprising a V _H polypeptide of SEQ ID NO: 726 and a V _L polypeptide of SEQ ID NO: 728;
(lxiv) comprising a V _H polypeptide of SEQ ID NO: 736 and a V _L polypeptide of SEQ ID NO: 738;
(lxv) comprising a V _H polypeptide of SEQ ID NO: 746 and a V _L polypeptide of SEQ ID NO: 748;
(lxvi) comprising a V _H polypeptide of SEQ ID NO: 756 and a V _L polypeptide of SEQ ID NO: 758;
(lxvii) comprising a V _H polypeptide of SEQ ID NO: 766 and a V _L polypeptide of SEQ ID NO: 768;
(lxviii) comprising a V _H polypeptide of SEQ ID NO: 776 and a V _L polypeptide of SEQ ID NO: 778;
(lxix) comprising a V _H polypeptide of SEQ ID NO: 786 and a V _L polypeptide of SEQ ID NO: 788;
(lxx) comprising a V _H polypeptide of SEQ ID NO: 796 and a V _L polypeptide of SEQ ID NO: 798;
(lxxxi) those comprising the V _H polypeptide of SEQ ID NO: 806 and the V _L polypeptide of SEQ ID NO: 808; and (lxxii) those comprising the V _H polypeptide of SEQ ID NO: 816 and the V _L polypeptide of SEQ ID NO: 818. The antibody-drug conjugate of any one of the preceding claims, wherein the antibody-drug conjugate possesses at least 90%, 95%, or 100% sequence identity with said antibody comprising: ADC). Any of the preceding claims, wherein the ADC comprises an anti-human VISTA antibody or antibody fragment, and wherein the anti-VISTA antibody or antibody fragment comprises the same variable region as one of VSTB92, VSTB56, VSTB95, VSTB103, and VSTB66. Antibody drug conjugate (ADC), use, medicament, composition, method according to item 1. The ADC optionally comprises an anti-human VISTA antibody or antibody fragment having the CDR or variable sequences of the antibody of FIG. Antibody drug conjugate (ADC), use, medicament, composition according to any one of the preceding claims, comprising a human IgG2 kappa backbone with V234A/G237A/P238S/H268A/V309L/A330S/P331S silencing mutations , or method. The ADC optionally comprises an anti-human VISTA antibody or antibody fragment having the CDR or variable sequences of the antibody of FIG. An antibody drug conjugate (ADC), use, or method according to any one of the preceding claims, comprising a human IgG1/kappa backbone with L234A/L235A silencing mutations. Any of the preceding claims, wherein said glucocorticosteroid agonist or linker conjugate is conjugated via its interchain disulfide to an antibody or antibody fragment, optionally an anti-human VISTA antibody or antibody fragment. ADC according to item 1. A pharmaceutical composition comprising a therapeutically effective amount of at least one antibody drug conjugate (ADC) or steroid agonist or steroid linker according to any one of the preceding claims and a pharmaceutically acceptable carrier. 95. A composition according to claim 94, optionally administrable via intravenous, intramuscular, intrathecal, or subcutaneous injection routes. 96. A composition according to claim 94 or 95, which is administrable subcutaneously. Glucocortico according to any one of the preceding claims, providing subcutaneous administration selected from the group consisting of syringes, injection devices, infusion pumps, injector pens, needleless devices, autoinjectors, and subcutaneous patch delivery systems. A device comprising a steroid agonist, linker conjugate, ADC, composition, or drug. 98. The device of claim 97, wherein a fixed dose of the glucocorticoid receptor agonist or functional derivative thereof is delivered to the patient. 99. A kit comprising the device of claim 97 or 98, further comprising instructions informing the patient of the method and dosing regimen for administering the ADC composition contained therein. A method of treatment and/or prevention, comprising administering to a patient in need of treatment and/or prevention at least one antibody drug conjugate (ADC) or steroid or composition according to any one of the preceding claims. 3. Administering the composition, wherein said composition may be present in a device according to any one of the preceding claims. In human subjects, for the treatment of allergy, autoimmunity, transplantation, gene therapy, inflammation, GVHD, or sepsis, or to treat or treat inflammatory, autoimmune, or allergic side effects associated with any of the foregoing conditions. 101. The method of claim 100, used for prophylaxis. 102. The method of claim 100 or 101, wherein the inflammation is associated with cancer or an infection, optionally a viral or bacterial infection. The patient has rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, sweat gland abscess, uveitis, Behcet's disease, spinal cord 102. The method of claim 100 or 101, having a condition selected from arthropathy, or psoriasis. The patient has one or more of the following:
(i) a chronic, acute, episodic allergy, inflammation, or inflammatory condition, e.g., chronic, acute, episodic, remitting/relapsing;
(ii) a condition that can be effectively treated only temporarily with high doses of steroids, optionally for which the patient is being treated or is being treated with high doses of steroids; polymyalgia and/or giant cell arteritis,
(iii) Conditions with comorbidities that limit steroid use, optionally diabetes, nonalcoholic steatohepatitis (NASH), morbid obesity, avascular necrosis/osteonecrosis (AVN), glaucoma, steroid-induced high blood pressure, severe skin fragility, and/or osteoarthritis;
(iv) conditions for which safe long-term therapeutic agents are available, but where induction for several months with high doses of steroids is desired; optionally, induction for several months with high doses of steroids is therapeutic; AAV, polymyositis, dermatomyositis, lupus, inflammatory lung disease, autoimmune hepatitis, inflammatory bowel disease, immune thrombocytopenia, autoimmune hemolytic anemia, gout patients,
(v) skin conditions requiring short-term/long-term treatment, optionally conditions of uncertain treatment or duration, and/or conditions for which there are no effective alternatives to steroid administration, optionally Stevens Johnson, other severe drug eruption conditions, conditions with extensive contact dermatitis, other severe immune-related skin conditions such as PG, LCV, erythroderma, and the like;
(vi) conditions treated with high-dose corticosteroids for flare-ups/relapses, optionally COPD, asthma, lupus, gout, pseudogout;
(vii) immune-related neurological diseases such as small fiber neuropathy, MS (subset), chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, and the like;
(viii) optionally hematology/oncology indications for which high doses of steroids are therapeutically warranted or beneficial;
(ix) ophthalmological conditions, optionally uveitis, iritis, scleritis, and the like;
(x) conditions associated with persistent or very long-term adrenal insufficiency or secondary adrenal insufficiency, optionally an iatrogenic Addison's disease crisis;
(xi) conditions often treated with long-term, low-dose steroids, optionally lupus, RA, psA, vasculitis, and the like; and (xii) pregnant/lactating women, pediatric patients, optionally A method according to any one of the preceding claims, optionally including special classes of patients, such as pediatric patients with growth disorders or cataracts. 12. A method, medicament, or use according to any preceding claim, wherein the patient is further treated with another active agent. The patient has an immunoinhibitory antibody or fusion protein targeting one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA; and/or further treated with an immunomodulatory antibody or fusion protein selected from agonist antibodies or fusion proteins targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28, or ICOS. A method, medicament or use according to any one of the claims. Immune cells from a patient or donor are contacted with an ADC or steroid according to any one of the preceding claims, and then a patient in need thereof, e.g. one of the conditions identified in the preceding claims, Ex vivo use of an ADC or steroid according to any one of the preceding claims, injected into a patient having one or more of the following. 6. ADC according to any one of the preceding claims, wherein the linker is a positively, negatively or neutrally charged cleavable peptide, optionally esterase cleavable. ADC according to any one of the preceding claims, wherein the drug-antibody ratio is in the range of 1-12:1 or 1-10:1. The ADC of any one of the preceding claims, wherein the drug-antibody ratio is in the range of 2-8:1, 4-8:1, or 6-8:1. The drug-antibody ratio is 4:1 (n = 4), 6:1 (n = 6), 8:1 (n = 8), 10:1 (n = 10), 12:1 (n = 12) or n is 12 or more and ranges from 12 to 50. Among activated or non-activated monocytes, myeloid cells, B cells, NK cells, T cells, CD4 T cells, CD8 T cells, Tregs, eosinophils, dendritic cells, mast cells, macrophages, and neutrophils An ADC according to any one of the preceding claims, internalizing one or more of the following. An ADC according to any one of the preceding claims, which does not appreciably internalize activated or non-activated B cells. When administered to a subject in need of treatment, the glucocorticoid receptor agonist promotes efficacy and/or enhances efficacy compared to the same dose of anti-inflammatory agent administered in naked (unconjugated) form. An ADC according to any one of the preceding claims, which reduces harmful side effects associated with. An antibody drug conjugate (ADC) according to any one of the preceding claims, wherein the glucocorticoid receptor agonist is conjugated to the antibody or antigen binding fragment via the interchain disulfide. An antibody drug conjugate (ADC) according to any one of the preceding claims, comprising an esterase sensitive linker. Any one of the preceding claims, wherein the cleavable linker is susceptible to one or more of acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Antibody drug conjugate (ADC) as described in Section. of the preceding claims, comprising a non-cleavable linker that is substantially resistant to one or more of acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Antibody drug conjugate (ADC) according to any one of the above. An antibody drug conjugate (ADC) according to any one of the preceding claims, wherein the anti-VISTA antigen binding fragment comprised in the ADC comprises a Fab, F(ab')2, or scFv antibody fragment. A method of treatment and/or prevention comprising administering to a patient in need of treatment and/or prevention at least one antibody drug conjugate (ADC) or composition, said composition comprising: The method may be present in a device according to any one of the preceding clauses. In human subjects, for the treatment of allergy, autoimmunity, transplantation, gene therapy, inflammation, GVHD, or sepsis, or to treat or treat inflammatory, autoimmune, or allergic side effects associated with any of the foregoing conditions. 121. The method of claim 120, used for prophylaxis. The patient has rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, sweat gland abscess, uveitis, Behcet's disease, spinal cord 122. The method of claim 120 or 121, having a condition selected from arthropathy, or psoriasis. The patient has one or more of the following:
(i) a condition that can be effectively treated only primarily with high doses of steroids, optionally where the patient is being treated with or is being treated with high doses of steroids; polymyalgia and/or giant cell arteritis,
(ii) Conditions with comorbidities that limit steroid use, optionally diabetes, nonalcoholic steatohepatitis (NASH), morbid obesity, avascular necrosis/osteonecrosis (AVN), glaucoma, steroid-induced high blood pressure, severe skin fragility, and/or osteoarthritis;
(iii) conditions for which safe long-term therapeutic agents are available, but where induction for several months with high doses of steroids is desired; optionally, induction for several months with high doses of steroids is therapeutic; AAV, polymyositis, dermatomyositis, lupus, inflammatory lung disease, autoimmune hepatitis, inflammatory bowel disease, immune thrombocytopenia, autoimmune hemolytic anemia, gout patients,
(iv) skin conditions requiring short-term/long-term treatment, optionally conditions of uncertain treatment or duration, and/or conditions for which there are no effective alternatives to steroid administration, optionally Stevens; Johnson, other severe drug eruption conditions, conditions with extensive contact dermatitis, other severe immune-related skin conditions such as PG, LCV, erythroderma, and the like;
(v) conditions treated with high-dose corticosteroids for flare-ups/relapses, optionally COPD, asthma, lupus, gout, pseudogout;
(vi) immune-related neurological diseases such as small fiber neuropathy, MS (subset), chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, and the like;
(vii) optionally, hematology/oncology indications for which high doses of steroids are therapeutically warranted or beneficial;
(viii) ophthalmological conditions, optionally uveitis, iritis, scleritis, and the like;
(ix) conditions associated with persistent or very long-term adrenal insufficiency or secondary adrenal insufficiency, optionally an iatrogenic Addison's disease crisis;
(x) Conditions often treated with long-term, low-dose steroids, optionally lupus, RA, psA, vasculitis, and the like; and (xi) Pregnant/lactating women, pediatric patients, optionally 123. A method according to any one of claims 120 to 122, optionally comprising special classes of patients, such as pediatric patients with growth disorders or cataracts. 124. The method of any one of claims 120-123, wherein the patient is further treated with another active agent. The patient has an immunoinhibitory antibody or fusion protein targeting one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA; and/or further treated with an immunomodulatory antibody or fusion protein selected from agonist antibodies or fusion proteins targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28, or ICOS. The method according to any one of paragraphs 120 to 124. for the treatment or prevention of acute or chronic inflammation and associated autoimmune and inflammatory indications, said conditions optionally including severe asthma, giant cell arteritis, ANKA vasculitis, and IBD. 126. The method according to any one of claims 120 to 125, comprising (colitis and Crohn's disease). Rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, sweat gland abscess, uveitis, Behcet's disease, spondyloarthropathy, or 127. A method according to any one of claims 115 to 126, for the treatment or prevention of a condition selected from psoriasis. in one or more of T cells, CD4 T cells, CD8 T cells, Tregs, NK cells, B cells, neutrophils, monocytes, myeloid cells, dendritic cells, eosinophils, mast cells, and macrophages A method for effecting internalization of glucocorticosteroids into a subject, comprising administering to a subject an ADC according to any one of the preceding claims, or contacting cells obtained from the subject ex vivo therewith. The method comprising: achieved ex vivo and containing immune cells or B cells, T cells, CD4 T cells, CD8 T cells, Tregs, NK cells, neutrophils, monocytes, myeloid cells, dendritic cells, eosinophils, obesity A purified or enriched composition comprising one or more specific types of immune cells selected from cells and macrophages is contacted ex vivo with an ADC according to any one of the preceding claims, and then 129. The method of claim 128, wherein the method is introduced into a patient in need thereof. Inflammation, autoimmunity, involving any one or more of B cells, T cells, Tregs, NK cells, neutrophils, monocytes, myeloid cells, dendritic cells, eosinophils, mast cells, and macrophages; or a method for treating an allergic condition, said method comprising administering to a subject in need of treatment an ADC according to any one of the preceding claims. A glucocorticoid agonist, composition containing, or method of use according to any one of the preceding claims, which does not consist of the formula:

the glucocorticoid agonist compound does not consist of a compound shown in FIG. 9 and/or having the following structure:

During the ceremony,
R=H, or R=

or R=

or R=

or R=

or R=

or R=

or R=

A glucocorticoid agonist, composition containing, or method of use according to any one of the preceding claims.

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